Up-converting reporters for biological and other assays using laser excitation techniques

The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

BACKGROUND OF THE INVENTION 
The invention relates generally to detectable labels and compositions 
useful in assay methods for detecting soluble, suspended, or particulate 
substances or analytes such as proteins, carbohydrates, nucleic acids, 
bacteria, viruses, and eukaryotic cells and more specifically relates to 
compositions and methods that include luminescent (phosphorescent or 
fluorescent) labels. 
Methods for detecting specific macromolecular species, such as proteins, 
drugs, and polynucleotides, have proven to be very valuable analytical 
techniques in biology and medicine, particularly for characterizing the 
molecular composition of normal and abnormal tissue samples and genetic 
material. Many different types of such detection methods are widely used 
in biomedical research and clinical laboratory medicine. Examples of such 
detection methods include: immunoassays, immunochemical staining for 
microscopy, fluorescence-activated cell sorting (FACS), nucleic acid 
hybridization, water sampling, air sampling, and others. 
Typically, a detection method employs at least one analytical reagent that 
binds to a specific target macromolecular species and produces a 
detectable signal. These analytical reagents typically have two 
components: (1) a probe macromolecule, for example, an antibody or 
oligonucleotide, that can bind a target macromolecule with a high degree 
of specificity and affinity, and (2) a detectable label, such as a 
radioisotope or covalently-linked fluorescent dye molecule. In general, 
the binding properties of the probe macromolecule define the specificity 
of the detection method, and the detectability of the associated label 
determines the sensitivity of the detection method. The sensitivity of 
detection is in turn related to both the type of label employed and the 
quality and type of equipment available to detect it. 
For example, radioimmunoassays (RIA) have been among the most sensitive and 
specific analytical methods used for detecting and quantitating biological 
macromolecules. Radioimmunoassay techniques have been used to detect and 
measure minute quantities of specific analytes, such as polypeptides, 
drugs, steroid hormones, polynucleotides, metabolites, and tumor markers, 
in biological samples. Radioimmunoassay methods employ immunoglobulins 
labeled with one or more radioisotopes as the analytical reagent. 
Radiation (.alpha., .beta., or .gamma.) produced by decay of the attached 
radioisotope label serves as the signal which can be detected and 
quantitated by various radiometric methods. 
Radioisotopic labels possess several advantages, such as: very high 
sensitivity of detection, very low background signal, and accurate 
measurement with precision radiometric instruments (scintillation and 
gamma counters) or with inexpensive and sensitive autoradiographic 
techniques. However, radioisotopic labels also have several disadvantages, 
such as: potential health hazards, difficulty in disposal, special 
licensing requirements, and instability (radioactive decay and 
radiolysis). Further, the fact that radioisotopic labels typically do not 
produce a strong (i.e., non-Cerenkov) signal in the ultraviolet, infrared, 
or visible portions of the electromagnetic spectrum makes radioisotopes 
generally unsuitable as labels for applications, such as microscopy, image 
spectroscopy, and flow cytometry, that employ optical methods for 
detection. 
For these and other reasons, the fields of clinical chemistry, water and 
air monitoring, and biomedical research have sought alternative detectable 
labels that do not require radioisotopes. Examples of such non-radioactive 
labels include: (1) enzymes that catalyze conversion of a chromogenic 
substrate to an insoluble, colored product (e.g., alkaline phosphatase, 
.beta.-galactosidase, horseradish peroxidase) or catalyze a reaction that 
yields a fluorescent or luminescent product (e.g., luciferase) (Beck and 
Koster (1990) Anal. Chem. 62: 2258; Durrant, I. (1990) Nature 346: 297; 
Analytical Applications of Bioluminescence and Chemiluminescence (1984) 
Kricka et al. (Eds.) Academic Press, London), and (2) direct fluorescent 
labels (e.g., fluorescein isothiocyanate, rhodamine, Cascade blue), which 
absorb electromagnetic energy in a particular absorption wavelength 
spectrum and subsequently emit visible light at one or more longer (i.e., 
less energetic) wavelengths. 
Using enzymes and phosphorescent/fluorescent or colorimetric detectable 
labels offers the significant advantage of signal amplification, since a 
single enzyme molecule typically has a persistent capacity to catalyze the 
transformation of a chromogenic substrate into detectable product. With 
appropriate reaction conditions and incubation time, a single enzyme 
molecule can produce a large amount of product, and hence yield 
considerable signal amplification. However, detection methods that employ 
enzymes as labels disadvantageously require additional procedures and 
reagents in order to provide a proper concentration of substrate under 
conditions suitable for the production and detection of the colored 
product. Further, detection methods that rely on enzyme labels typically 
require prolonged time intervals for generating detectable quantities of 
product, and also generate an insoluble product that is not attached to 
the probe molecule. 
An additional disadvantage of enzyme labels is the difficulty of detecting 
multiple target species with enzyme-labeled probes. It is problematic to 
optimize reaction conditions and development time(s) for two or more 
discrete enzyme label species and, moreover, there is often considerable 
spectral overlap in the chromophore end products which makes 
discrimination of the reaction products difficult. 
Fluorescent labels do not offer the signal amplification advantage of 
enzyme labels, nonetheless, fluorescent labels possess significant 
advantages which have resulted in their widespread adoption in 
immunocytochemistry. Fluorescent labels typically are small organic dye 
molecules, such as fluorescein, Texas Red, or rhodamine, which can be 
readily conjugated to probe molecules, such as immunoglobulins or Staph. 
aureus Protein A. The fluorescent molecules (fluorophores) can be detected 
by illumination with light of an appropriate excitation frequency and the 
resultant spectral emissions can be detected by electro-optical sensors or 
light microscopy. 
A wide variety of fluorescent dyes are available and offer a selection of 
excitation and emission spectra. It is possible to select fluorophores 
having emission spectra that are sufficiently different so as to permit 
multitarget detection and discrimination with multiple probes, wherein 
each probe species is linked to a different fluorophore. Because the 
spectra of fluorophores can be discriminated on the basis of both narrow 
band excitation and selective detection of emission spectra, two or more 
distinct target species can be detected and resolved (Titus et al. (1982) 
J. Immunol. Methods 50: 193; Nederlof et al. (1989) Cytometry 10: 20; 
Ploem, J. S. (1971) Ann. N.Y. Acad. Sci. 177: 414). 
Unfortunately, detection methods which employ fluorescent labels are of 
limited sensitivity for a variety of reasons. First, with conventional 
fluorophores it is difficult to discriminate specific fluorescent signals 
from nonspecific background signals. Most common fluorophores are aromatic 
organic molecules which have broad absorption and emission spectra, with 
the emission maximum red-shifted 50-100 nm to a longer wavelength than the 
excitation (i.e., absorption) wavelength. Typically, both the absorption 
and emission bands are located in the UV/visible portion of the spectrum. 
Further, the lifetime of the fluorescence emission is usually short, on 
the order of 1 to 100 ns. Unfortunately, these general characteristics of 
organic dye fluorescence are also applicable to background signals which 
are contributed by other reagents (e.g., fixative or serum), or 
autofluorescence or the sample itself (Jongkind et al. (1982) Exp. Cell 
Res. 138: 409; Aubin, J. E. (1979) J. Histochem. Cytochem. 27: 36). 
Autofluorescence of optical lenses and reflected excitation light are 
additional sources of background noise in the visible spectrum (Beverloo 
et al. (1991) Cytometry 11: 784; Beverloo et al. (1992) Cytometry 13: 
561). Therefore, the limit of detection of specific fluorescent signal 
from typical fluorophores is limited by the significant background noise 
contributed by nonspecific fluorescence and reflected excitation light. 
A second problem of organic dye fluorophores that limits sensitivity is 
photolytic decomposition of the dye molecule (i.e., photobleaching). Thus, 
even in situations where background noise is relatively low, it is often 
not possible to integrate a weak fluorescent signal over a long detection 
time, since the dye molecules decompose as a function of incident 
irradiation in the UV and near-UV bands. 
However, because fluorescent labels are attractive for various 
applications, several alternative fluorophores having advantageous 
properties for sensitive detection have been proposed. One approach has 
been to employ organic dyes comprising a phycobiliprotein acceptor 
molecule dye that emits in the far red or near infrared region of the 
spectrum where nonspecific fluorescent noise is reduced. Phycobiliproteins 
are used in conjunction with accessory molecules that effect a large 
Stokes shift via energy transfer mechanisms (U.S. Pat. No. 4,666,862; Oi 
et al. (1982) J. Cell. Biol. 93: 891). Phycobiliprotein labels reduce the 
degree of spectral overlap between excitation frequencies and emission 
frequencies. An alternative approach has been to use cyanine dyes which 
absorb in the yellow or red region and emit in the red or far red where 
autofluorescence is reduced (Mujumbar et al. (1989) Cytometry 19: 11). 
However, with both the phycobiliproteins and the cyanine dyes the emission 
frequencies are red-shifted (i.e., frequency downshifted) and emission 
lifetimes are short, therefore background autofluorescence is not 
completely eliminated as a noise source. More importantly perhaps, 
phycobiliproteins and cyanine dyes possess several distinct disadvantages: 
(1) emission in the red, far red, and near infrared region is not 
well-suited for detection by the human eye, hampering the use of 
phycobiliprotein and cyanine labels in optical fluorescence microscopy, 
(2) cyanines, phycobiliproteins, and the coupled accessory molecules 
(e.g., Azure A) are organic molecules susceptible to photobleaching and 
undergoing undesirable chemical interactions with other reagents, and (3) 
emitted radiation is down-converted, i.e., of longer wavelength(s) than 
the absorbed excitation radiation. For example, Azure A absorbs at 632 nm 
and emits at 645 nm, and allophycocyanin absorbs at 645 nm and emits at 
655 nm, and therefore autofluorescence and background noise from scattered 
excitation light is not eliminated. 
Another alternative class of fluorophore that has been proposed are the 
down-converting luminescent lanthanide chelates (Soini and Lovgren (1987) 
CRC Crit. Rev. Anal. Chem. 18: 105; Leif et al. (1977) Clin. Chem. 23: 
1492; Soini and Hemmila (1979) Clin. Chem. 25: 353; Seveus et al. (1992) 
Cytometry 13: 329). Down-converting lanthanide chelates are inorganic 
phosphors which possess a large downward Stokes shift (i.e., emission 
maxima is typically at least 100 nm greater than absorption maxima) which 
aids in the discrimination of signal from scattered excitation light. 
Lanthanide phosphors possess emission lifetimes that are sufficiently long 
(i.e., greater than 1 .mu.s) to permit their use in time-gated detection 
methods which can reduce, but not totally eliminate, noise caused by 
shorter-lived autofluorescence and scattered excitation light. Further, 
lanthanide phosphors possess narrow-band emission, which facilitates 
wavelength discrimination against background noise and scattered 
excitation light, particularly when a laser excitation source is utilized 
(Reichstein et al. (1988) Anal. Chem. 60: 1069). Recently, 
enzyme-amplified lanthanide luminescence using down-converting lanthanide 
chelates has been proposed as a fluorescent labeling technique 
(Evangelista et al. (1991) Anal. Biochem. 197: 213; Gudgin-Templeton et 
al. (1991) Clin Chem. 37: 1506). 
Until recently, down-converting lanthanide phosphors have had the 
significant disadvantage that their quantum efficiency in aqueous 
(oxygenated) solutions is so low as to render them unsuitable for 
cytochemical staining. Beverloo et al. (op.cit.) have described a 
particular down-converting lanthanide phosphor (yttrium oxysulfide 
activated with europium) that produces a signal in aqueous solutions which 
can be detected by time-resolved methods. Seveus et al. (op.cit.) have 
used down-converting europium chelates in conjunction with time-resolved 
fluorescence microscopy to reject the signal from prompt fluorescence and 
thereby reduce autofluorescence. Tanke et al. (U.S. Pat. No. 5,043,265) 
report down-converting phosphor particles as labels for immunoglobulins 
and polynucleotides. 
However, the down-converting lanthanide phosphor of Beverloo et al. and the 
europium chelate of Seveus et al. require excitation wavelength maxima 
that are in the ultraviolet range, and thus produce significant sample 
autofluorescence and background noise (e.g., serum and/or fixative 
fluorescence, excitation light scattering and refraction, etc.) that must 
be rejected (e.g., by filters or time-gated signal rejection). Further, 
excitation with ultraviolet irradiation damages nucleic acids and other 
biological macromolecules, posing serious problems for immunocytochemical 
applications where it is desirable to preserve the viability of living 
cells and retain cellular structures (e.g., FACS, cyto-architectural 
microscopy). 
Laser scanning fluorescence microscopy has been used for two-photon 
excitation of a UV-excitable fluorescent organic dye, Hoechst 33258, using 
a stream of strongly focused laser pulses (Denk et al. (1990) Science 248: 
73). The organic fluorphore used by Denk et al. was significantly 
photobleached by the intense, highly focused laser light during the course 
of imaging. Motsenbocker et al. (EP 476 556) describes a method to 
increase luminol chemiluminescence by adding a dye catalyst that absorbs 
long wavelength radiation (deep red light) and subsequently reacts with 
molecular oxygen to generate an oxidant which can itself react with 
luminol and produce oxidized luminol which emits blue light. Gavrilovic 
(U.S. Pat. No. 5,166,948) discloses a method and apparatus for optical 
pumping of infrared pump light to a visible or ultraviolet emission light 
having a wavelength shorter than the pump light (i.e., up-converted 
emission). 
Thus, there exists a significant need in the art for labels and detection 
methods that permit sensitive optical and/or spectroscopic detection of 
specific label signal(s) with essentially total rejection of nonspecific 
background noise, and which are compatible with intact viable cells and 
aqueous or airborne environments. 
The references discussed herein are provided solely for their disclosure 
prior to the filing date of the present application. Nothing herein is to 
be construed as an admission that the inventors are not entitled to 
antedate such disclosure by virtue of prior invention. 
SUMMARY OF THE INVENTION 
The present invention provides labels, detection methods, and detection 
apparatus which permit ultrasensitive detection of cells, biological 
macromolecules, and other analytes, which can be used for multiple target 
detection and target discrimination. The up-converting labels of the 
invention permit essentially total rejection of non-specific background 
autofluorescence and are characterized by excitation and emitted 
wavelengths that are typically in the infrared or visible portions of the 
spectrum, respectively, and thus avoid the potentially damaging effects of 
ultraviolet radiation. The up-converting labels of the invention convert 
long-wavelength excitation radiation (e.g., near-IR) to emitted radiation 
at about one-half to one-third the wavelength of the excitation 
wavelength. Since background fluorescence in the visible range is 
negligible if near-IR excitation wavelengths are used, the use of 
up-converting labels provides essentially background-free detection of 
signal. 
In brief, the invention provides the use of luminescent materials that are 
capable of multiphoton excitation and have upshifted emission spectra. In 
one embodiment of the invention, up-converting phosphors (i.e., which 
absorb multiple photons in a low frequency band and emit in a higher 
frequency band) are used as labels which can be linked to one or more 
probes, such as an immunoglobulin, polynucleotide, streptavidin, Protein 
A, receptor ligand, or other probe molecule. In an another embodiment, 
up-converting organic dyes serve as the label. The organic dye labels and 
phosphor labels of the invention are highly compatible with automated 
diagnostic testing, microscopic imaging applications, and coded particle 
detection, among many other applications. 
The nature of the invention provides considerable flexibility in the 
apparatus for carrying out the methods. As a general matter, the 
excitation source may be any convenient light source, including 
inexpensive near-infrared laser diodes or light-emitting diodes (LEDs), 
and the detector may be any convenient detector, such as a photodiode. In 
the case of a single reporter, the apparatus includes a laser diode 
capable of emitting light at one or more wavelengths in the reporter's 
excitation band and a detector that is sensitive to at least some 
wavelengths in the reporter's emission band. The laser light is preferably 
focused to a small region in the sample, and light emanating from that 
region is collected and directed to the detector. An electrical signal 
representing the intensity of light in the emission band provides a 
measure of the amount of reporter present. Depending on the detector's 
spectral response, it may be necessary to provide a filter to block the 
excitation light. 
Simultaneous detection of multiple reporters is possible, at least where 
the reporters have different excitation bands or different emission bands. 
Where the excitation bands differ, multiple laser diodes emitting at 
respective appropriate wavelengths are combined using a wavelength 
division multiplexer or other suitable techniques, such as frequency 
labeling, frequency modulation, and lock-in detector device. If the 
emission bands are different (whether or not the excitation bands are 
different), light in the different emission bands is separated and sent to 
multiple detectors. If the emission bands overlap, a single detector may 
be used, but other detection techniques are used. One example is to use 
time multiplexing techniques so that only one reporter is emitting at a 
given time. Alternatively, the different laser diodes can be modulated at 
different characteristic frequencies and lock-in detection performed. 
Detection methods and detection apparatus of the present invention enable 
the ultrasensitive detection of up-converting phosphors and up-converting 
organic dyes by exploiting what is essentially the total absence of 
background noise (e.g., autofluorescence, serum/fixative fluorescence, 
excitation light scatter) that are advantageous characteristics of 
up-converting labels. Some embodiments of the invention utilize time-gated 
detection and/or wavelength-gated detection for optimizing detection 
sensitivity, discriminating multiple samples, and/or detecting multiple 
probes on a single sample. Phase-sensitive detection can also be used to 
provide discrimination between signal(s) attributable to an up-converting 
phosphor and background noise (e.g. autofluorescence) which has a 
different phase shift. 
Up-converting organic dyes, such as red-absorbing dyes, also can be used in 
an alternate embodiment that converts the photons absorbed by the dye into 
a transient voltage that can be measured using electrodes and conventional 
electronic circuitry. After having undergone two-photon absorption the dye 
is ionized by additional photons from the light source (e.g., a laser) 
leading to short-lived molecular ions whose presence can be detected and 
quantified by measuring the transient photoconductivity following the 
excitation irradiation. In this embodiment, resonant multiphoton 
ionization is used to provide a quantitative measurement of the number 
and/or concentration of dye molecules in a sample. Furthermore, 
essentially all photoions formed in the irradiated sample contribute to 
the signal, whereas photons are emitted isotopically and only a fraction 
can be collected using optics. Measurement of the transient photocurrent 
effectively transfers the conversion of photons into an electronic signal 
that is readily measured with relatively simple and inexpensive sensors 
such as electrodes. 
In some embodiments, the present invention utilizes one or more optical 
laser sources for generating excitation illumination of one or more 
discrete frequency(ies). In certain variations of the invention, laser 
irradiation of an up-converting label can modify the immediate molecular 
environment through laser-induced photochemical processes involving either 
direct absorption or energy transfer; such spatially-controlled deposition 
of energy can be used to produce localized damage and/or to probe the 
chemical environment of a defined location. In such embodiments, the 
up-converting label can preferably act as a photophysical catalyst. 
The invention provides methods for producing targeted damage (e.g., 
catalysis) in chemical or biological materials, wherein a probe is 
employed to localize a linked up-converting label to a position near a 
targeted biological structure that is bound by the probe. The localized 
up-converting label is excited by one or more excitation wavelengths and 
emits at a shorter wavelength which may be directly cytotoxic or genotoxic 
(e.g., by producing free radicals such as superoxide, and/or by generating 
thymine-thymine dimers), or which may induce a local photolytic chemical 
reaction to produce reactive chemical species in the immediate vicinity of 
the label, and hence in the vicinity of the targeted biological material. 
Thus, targeting probes labeled with one or more up-converting labels 
(e.g., an up-converting inorganic phosphor) may be used to produce 
targeted damage to biological structures, such as cells, tissues, 
neoplasms, vasculature, or other anatomical or histological structures. 
Embodiments of the present invention also include up-converting phosphors 
which can also be excited by an electron beam or other beam of energetic 
radiation of sufficient energy and are cathodoluminescent. Such 
electron-stimulated labels afford novel advantages in eliminating 
background in ultrasensitive biomolecule detection methods. Typically, 
stimulation of the up-converting phosphor with at least two electrons is 
employed to generate a visible-light or UV band emission. 
The invention also provides for the simultaneous detection of multiple 
target species by exploiting the multiphoton excitation and subsequent 
background-free fluorescence detection of several up-converting phosphors 
or up-converting dyes. In one embodiment, several phosphors/dyes are 
selected which have overlapping absorption bands which allow simultaneous 
excitation at one wavelength (or in a narrow bandwidth), but which vary in 
emission characteristics such that each probe-label species is endowed 
with a distinguishable fluorescent "fingerprint." By using various methods 
and devices, the presence and concentration of each of the phosphors or 
dyes can be determined. 
The invention also provides biochemical assay methods for determining the 
presence and concentration of one or more analytes, typically in solution. 
The assay methods employ compositions of probes labeled with up-converting 
phosphors and/or up-converting dyes and apparatus for magnetically and/or 
optically trapping particles that comprise the analyte and the labeled 
probe. In one embodiment, a sandwich assay is performed, wherein an 
immobilized probe, immobilized on a particle, binds to a predetermined 
analyte, producing an immobilization of the bound analyte on the particle; 
a second probe, labeled with an up-converting label can then bind to the 
bound analyte to produce a bound sandwich complex containing an 
up-converting label bound to a particle. By combining different 
probe-label combinations, particles of various sizes, colors, and/or 
shapes with distinct immobilized probe(s), and/or various excitation 
wavelengths, it is possible to perform multiple assays essentially 
simultaneously or contemporaneously. This multiplex advantage affords 
detection and quantitation of multiple analyte species in a single sample. 
The assay methods are also useful for monitoring the progress of a 
reaction, such as a physical, chemical, biochemical, or immunological 
reaction, including binding reactions. For example, the invention may be 
used to monitor the progress of ligand-binding reactions, polynucleotide 
hybridization reactions, including hybridization kinetics and 
thermodynamic stability of hybridized polynucleotides. 
The invention also provides methods, up-converting labels, and compositions 
of labeled binding reagents for performing fluorescence-activated cell 
sorting (FACS) by flow cytometry using excitation radiation that is in the 
infrared portion of the spectrum and does not significantly damage cells. 
This provides a significant advantage over present FACS methods which rely 
on excitation illumination in the ultraviolet portion of the spectrum, 
including wavelengths which are known to produce DNA lesions and damage 
cells. 
The invention also provides compositions comprising at least one 
fluorescent organic dye molecule attached to an inorganic up-converting 
phosphor. The fluorescent organic dye molecule is selected from the group 
consisting of: rhodamines, cyanines, xanthenes, acridines, oxazines, 
porphyrins, and phthalocyanines, and may optionally be complexed with a 
heavy metal. The fluorescent organic dye may be adsorbed to the inorganic 
up-converting phosphor crystal and/or may be covalently attached to a 
coated inorganic up-converting phosphor, a derivatized vitroceramic 
up-converting phosphor, or a microencapsulated inorganic up-converting 
phosphor. Frequently, covalent conjugation between the up-converting 
inorganic phosphor particles and proteins (e.g., avidin, immunoglobulin) 
can be accomplished with heterobifunctional crosslinkers.

DESCRIPTION OF SPECIFIC EMBODIMENTS 
Definitions 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods 
and materials are described. For purposes of the present invention, the 
following terms are defined below. 
As used herein, "label" refers to a chemical substituent that produces, 
under appropriate excitation conditions, a detectable optical signal. The 
optical signal produced by an excited label is typically electromagnetic 
radiation in the near-infrared, visible, or ultraviolet portions of the 
spectrum. The labels of the invention are up-converting labels, which 
means that the chemical substituent absorbs at least two photons at an 
excitation frequency and subsequently emits electromagnetic energy at an 
emission frequency higher than the excitation frequency. Thus, there is 
generally a significant Stokes shift between the original excitation 
frequency and the final emission frequency. A label is generally attached 
to a probe to serve as a reporter that indicates the presence and/or 
location of probe. The invention encompasses organic and inorganic 
up-converting labels, but preferably employs up-converting inorganic 
lanthanide phosphors as labels. Thus, a typical label of the invention is 
a submicron-size up-converting lanthanide phosphor particle. The label can 
alternatively comprise a lanthanide ion in a chelate or cage compound. 
As used herein, a "probe" refers to a binding component which binds 
preferentially to one or more targets (e.g, antigenic epitopes, 
polynucleotide sequences, macromolecular receptors) with an affinity 
sufficient to permit discrimination of labeled probe bound to target from 
nonspecifically bound labeled probe (i.e., background). Generally, the 
probe-target binding is a non-covalent interaction with a binding affinity 
(K.sub.D) of at least about 1.times.10.sup.6 M.sup.-1, preferably with at 
least about 1.times.10.sup.7 M.sup.-1, and more preferably with an 
affinity of at least about 1.times.10.sup.8 M.sup.-1 or greater. 
Antibodies typically have a binding affinity for cognate antigen of about 
1.times.10.sup.10 M.sup.-1 or more. For example but not limitation, probes 
of the invention include: antibodies, polypeptide hormones, 
polynucleotides, streptavidin, Staphlyococcus aureus protein A, receptor 
ligands (e.g., steroid or polypeptide hormones), leucine zipper 
polypeptides, lectins, antigens (polypeptide, carbohydrate, nucleic acid, 
and hapten epitopes), and others. 
As used herein, a "probe-label conjugate" and a "labeled probe" refer to a 
combination comprising a label attached to a probe. In certain 
embodiments, more than one label substituent may be attached to a probe. 
Alternatively, in some embodiments more than one probe may be attached to 
a label (e.g., multiple antibody molecules may be attached to a 
submicron-size inorganic up-converting phosphor bead). Various attachment 
chemistries can be employed to link a label to a probe, including, but not 
limited to, the formation of: covalent bonds, hydrogen bonds, ionic bonds, 
electrostatic interactions, and surface tension (phase boundary) 
interactions. Attachment of label can also involve incorporation of the 
label into or onto microspheres, microparticles, immunobeads, and 
superparamagnetic magnetic beads (Polysciences, Inc., Warrington, Pa.; 
Bangs Laboratories, Inc. 979 Keystone Way, Carmel, Ind. 46032). For 
example, inorganic up-converting phosphor particles can be encapsulated in 
microspheres that are composed of polymer material that is essentially 
transparent or translucent in the wavelength range(s) of the excitation 
and emitted electromagnetic radiation (U.S. Pat. No. 5,132,242, 
incorporated herein by reference). Such microspheres can be functionalized 
by surface derivatization with one or more reactive groups (e.g., 
carboxylate, amino, hydroxylate, or polyacrolein) for covalent attachment 
to a probe, such as a protein. Probe-label conjugates can also comprise a 
phosphor chelate. 
As used herein, the term "target" and "target analyte" refer to the 
object(s) that is/are assayed for by the methods of the invention. For 
example but not limitation, targets can comprise polypeptides (e.g., hGH, 
insulin, albumin), glycoproteins (e.g., immunoglobulins, thrombomodulin, 
.gamma.-glutamyltranspeptidase; Goodspeed et al. (1989) Gene 76: 1), 
lipoproteins, viruses, microorganisms (e.g., pathogenic bacteria, yeasts), 
polynucleotides (e.g., cellular genomic DNA, RNA in a fixed histological 
specimen for in situ hybridization, DNA or RNA immobilized on a nylon or 
nitrocellulose membrane, viral DNA or RNA in a tissue or biological 
fluid), and pharmaceuticals (i.e., prescribed or over-the-counter drugs 
listed in the Physicians Drug Reference and/or Merck Manual, or illegal 
substances such as intoxicants or anabolic steroids). 
As used herein, the term "antibody" refers to a protein consisting of one 
or more polypeptides substantially encoded by immunoglobulin genes. The 
recognized immunoglobulin genes include the kappa, lambda, alpha, gamma 
(IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4), delta, epsilon and mu 
constant region genes, as well as the myriad immunoglobulin variable 
region genes. Full-length immunoglobulin "light chains" (about 25 Kd or 
214 amino acids) are encoded by a variable region gene at the NH2-terminus 
(about 110 amino acids) and a kappa or lambda constant region gene at the 
COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or 
446 amino acids), are similarly encoded by a variable region gene (about 
116 amino acids) and one of the other aforementioned constant region 
genes, e.g., gamma (encoding about 330 amino acids). One form of 
immunoglobulin constitutes the basic structural unit of an antibody. This 
form is a tetramer and consists of two identical pairs of immunoglobulin 
chains, each pair having one light and one heavy chain. In each pair, the 
light and heavy chain variable regions are together responsible for 
binding to an antigen, and the constant regions are responsible for the 
antibody effector functions. In addition to antibodies, immunoglobulins 
may exist in a variety of other forms including, for example, Fv, Fab, and 
F(ab').sub.2, as well as bifunctional hybrid antibodies (e.g., 
Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains 
(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) 
and Bird et al., Science, 242, 423-426 (1988)). (See, generally, Hood et 
al., "Immunology", Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and 
Hood, Nature, 323, 15-16 (1986)). Thus, not all immunoglobulins are 
antibodies. (See, U.S. Ser. No. 07/634,278, which is incorporated herein 
by reference, and Co et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 
2869, which is incorporated herein by reference). 
As used herein, "probe polynucleotide" refers to a polynucleotide that 
specifically hybridizes to a predetermined target polynucleotide. For 
example but not limitation, a probe polynucleotide may be a portion of a 
cDNA corresponding to a particular mRNA sequence, a portion of a genomic 
clone, a synthetic oligonucleotide having sufficient sequence homology to 
a known target sequence (e.g., a telomere repeat TTAGGG or an Alu 
repetitive sequence) for specific hybridization, a transcribed RNA (e.g., 
from an SP6 cloning vector insert), or a polyamide nucleic acid (Nielsen 
et al. (1991) Science 254: 1497). Various target polynucleotides may be 
detected by hybridization of a labeled probe polynucleotide to the target 
sequence(s). For example but not limitation, target polynucleotides may 
be: genomic sequences (e.g., structural genes, chromosomal repeated 
sequences, regulatory sequences, etc.), RNA (e.g., mRNA, hnRNA, rRNA, 
etc.), pathogen sequences (e.g., viral or mycoplasmal DNA or RNA 
sequences), or transgene sequences. 
"Specific hybridization" is defined herein as the formation of hybrids 
between a probe polynucleotide and a target polynucleotide, wherein the 
probe polynucleotide preferentially hybridizes to the target DNA such 
that, for example, at least one discrete band can be identified on a 
Southern blot of DNA prepared from eukaryotic cells that contain the 
target polynucleotide sequence, and/or a probe polynucleotide in an intact 
nucleus localizes to a discrete chromosomal location characteristic of a 
unique or repetitive sequence. In some instances, a target sequence may be 
present in more than one target polynucleotide species (e.g., a particular 
target sequence may occur in multiple members of a gene family or in a 
known repetitive sequence). It is evident that optimal hybridization 
conditions will vary depending upon the sequence composition and length(s) 
of the targeting polynucleotide(s) and target(s), and the experimental 
method selected by the practitioner. Various guidelines may be used to 
select appropriate hybridization conditions (see, Maniatis et al., 
Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring 
Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume 152, 
Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San 
Diego, Calif., Dunn et al. (1989) J. Biol. Chem. 264: 13057 and Goodspeed 
et al. (1989) Gene 76: 1. 
As used herein, the term "label excitation wavelength" refers to an 
electromagnetic radiation wavelength that, when absorbed by an 
up-converting label, produces a detectable fluorescent emission from the 
up-converting label, wherein the fluorescent emission is of a shorter 
wavelength (i.e., higher frequency radiation) that the label excitation 
wavelength. As used herein, the term "label emission wavelength" refers to 
a wavelength that is emitted from an up-converting label subsequent to, or 
contemporaneously with, illumination of the up-converting label with one 
or more excitation wavelengths; label emission wavelengths of 
up-converting labels are shorter (i.e., higher frequency radiation) than 
the corresponding excitation wavelengths. Both label excitation 
wavelengths and label emission wavelengths are characteristic to 
individual up-converting label species, and are readily determined by 
performing simple excitation and emission scans. 
Invention Overview 
The subject invention encompasses fluorescent labels that are excited by an 
excitation wavelength and subsequently emit electromagnetic radiation at 
up-shifted frequencies (i.e., at higher frequencies than the excitation 
radiation). 
In accordance with the present invention, labels comprising up-converting 
inorganic phosphors and/or up-converting organic dyes are provided for 
various applications. The up-converting labels of the invention may be 
attached to one or more probe(s) to serve as a reporter (i.e., a 
detectable marker) of the location of the probe(s). The up-converting 
labels can be attached to various probes, such as antibodies, 
streptavidin, protein A, polypeptide ligands of cellular receptors, 
polynucleotide probes, drugs, antigens, toxins, and others. Attachment of 
the up-converting label to the probe can be accomplished using various 
linkage chemistries, depending upon the nature of the specific probe. For 
example but not limitation, microcrystalline up-converting lanthanide 
phosphor particles may be coated with a polycarboxylic acid (e.g., Additon 
XW 330, Hoechst, Frankfurt, Germany) during milling and various proteins 
(e.g., immunoglobulin, streptavidin or protein A) can be physically 
adsorbed to the surface of the phosphor particle (Beverloo et al. (1991) 
op.cit., which is incorporated herein by reference). Alternatively, 
various inorganic phosphor coating techniques can be employed including, 
but not limited to: spray drying, plasma deposition, and derivatization 
with functional groups (e.g., --COOH, --NH.sub.2, --CONH.sub.2) attached 
by a silane coupling agent to --SiOH moieties coated on the phosphor 
particle or incorporated into a vitroceramic phosphor particle comprising 
silicon oxide(s) and up-converting phosphor compositions. Vitroceramic 
phosphor particles can be aminated with, for example, 
aminopropyltriethoxysilane for the purpose of attaching amino groups to 
the vitroceramic surface on linker molecules, however other 
omega-functionalized silanes can be substituted to attach alternative 
functional groups. Probes, such as proteins or polynucleotides may then be 
directly attached to the vitroceramic phosphor by covalent linkage, for 
example through siloxane bonds or through carbon-carbon bonds to linker 
molecules (e.g., organofunctional silylating agents) that are covalently 
bonded to or adsorbed to the surface of a phosphor particle. Covalent 
conjugation between the up-converting inorganic phosphor particles and 
proteins (e.g., avidin, immunoglobulin) can be accomplished with 
homobifunctional, or preferably heterobifunctional, crosslinkers. For 
example, surface slianization of the phosphors with tri(ethoxy)thiopropyl 
silane leaves a phosphor surface with a thiol functionality to which a 
protein (e.g., antibody) or any compound containing a primary amine can be 
grafted using conventional N-succinimidyl(4-iodoacetyl)amino-benzoate 
(SIAB) chemistry (Weltman et al. (1983). Other silanization and 
cross-linking methods compatible with the inorganic phosphors may be used 
at the discretion of the practitioner. 
Microcrystalline up-converting phosphor particles are typically smaller 
than about 3 microns in diameter, preferably less than about 1 micron in 
diameter (i.e., submicron), and more preferably are 0.1 to 0.3 microns or 
less in diameter. It is generally most preferred that the phosphor 
particles are as small as possible while retaining sufficient quantum 
conversion efficiency to produce a detectable signal; however, for any 
particular application, the size of the phosphor particle(s) to be used 
should be selected at the discretion of the practitioner. For instance, 
some applications (e.g., detection of a non-abundant cell surface antigen) 
may require a highly sensitive phosphor label that need not be small but 
must have high conversion efficiency and/or absorption cross-section, 
while other applications (e.g., detection of an abundant nuclear antigen 
in a permeablized cell) may require a very small phosphor particle that 
can readily diffuse and penetrate subcellular structures, but which need 
not have high conversion efficiency. Therefore, the optimal size of 
inorganic phosphor particle is application dependent and is selected by 
the practitioner on the basis of quantum efficiency data for the various 
phosphors of the invention. Such conversion efficiency data may be 
obtained from available sources (e.g., handbooks and published references) 
or may be obtained by generating a standardization curve measuring quantum 
conversion efficiency as a function of particle size. In some 
applications, such as those requiring highly sensitive detection of small 
phosphor particles, infrared laser diodes are preferably selected as an 
excitation source. 
Although the properties of the up-converting phosphors will be described in 
detail in a later section, it is useful to outline the basic mechanisms 
involved. Up-conversion has been found to occur in certain materials 
containing rare-earth ions in certain crystal materials. For example, 
ytterbium and erbium act as an activator couple in a phosphor host 
material such as barium-yttrium-fluoride. The ytterbium ions act as the 
absorber, and transfer energy non-radiatively to excite the erbium ions. 
The emission is thus characteristic of the erbium ion's energy levels. 
Up-Converting Microcrystalline Phosphors 
Although the invention can be practiced with a variety of up-converting 
inorganic phosphors, it is believed that the preferred embodiment(s) 
employ one or more phosphors derived from one of several different 
phosphor host materials, each doped with at least one activator couple. 
Suitable phosphor host materials include: sodium yttrium fluoride 
(NaYF.sub.4), lanthanum fluoride (LaF.sub.3), lanthanum oxysulfide, 
yttrium oxysulfide, yttrium fluoride (YF.sub.3), yttrium gallate, yttrium 
aluminum garnet, gadolinium fluoride (GdF.sub.3), barium yttrium fluoride 
(BaYF.sub.5, BaY.sub.2 F.sub.S), and gadolinium oxysulfide. Suitable 
activator couples are selected from: ytterbium/erbium, ytterbium/thulium, 
and ytterbium/holmium. Other activator couples suitable for up-conversion 
may also be used. By combination of these host materials with the 
activator couples, at least three phosphors with at least three different 
emission spectra (red, green, and blue visible light) are provided. 
Generally, the absorber is ytterbium and the emitting center can be 
selected from: erbium, holmium, terbium, and thulium; however, other 
up-converting phosphors of the invention may contain other absorbers 
and/or emitters. The molar ratio of absorber: emitting center is typically 
at least about 1:1, more usually at least about 3:1 to 5:1, preferably at 
least about 8:1 to 10:1, more preferably at least about 11:1 to 20:1, and 
typically less than about 250:1, usually less than about 100:1, and more 
usually less than about 50:1 to 25:1, although various ratios may be 
selected by the practitioner on the basis of desired characteristics 
(e.g., chemical properties, manufacturing efficiency, absorption 
cross-section, excitation and emission wavelengths, quantum efficiency, or 
other considerations). The ratio(s) chosen will generally also depend upon 
the particular absorber-emitter couple(s) selected, and can be calculated 
from reference values in accordance with the desired characteristics. 
The optimum ratio of absorber (e.g., ytterbium) to the emitting center 
(e.g., erbium, thulium, or holmium) varies, depending upon the specific 
absorber/emitter couple. For example, the absorber:emitter ratio for Yb:Er 
couples is typically in the range of about 20:1 to about 100:1, whereas 
the absorber:emitter ratio for Yb:Tm and Yb:Ho couples is typically in the 
range of about 500:1 to about 2000:1. These different ratios are 
attributable to the different matching energy levels of the Er, Tm, or Ho 
with respect to the Yb level in the crystal. For most applications, 
up-converting phosphors may conveniently comprise about 10-30% Yb and 
either: about 1-2% Er, about 0.1-0.05% Ho, or about 0.1-0.05% Tm, although 
other formulations may be employed. 
Some embodiments of the invention employ inorganic phosphors that are 
optimally excited by infrared radiation of about 950 to 1000 nm, 
preferably about 960 to 980 nm. For example but not limitation, a 
microcrystalline inorganic phosphor of the formula YF.sub.3 :Yb.sub.0.10 
Er.sub.0.01 exhibits a luminescence intensity maximum at an excitation 
wavelength of about 980 nm. Inorganic phosphors of the invention typically 
have emission maxima that are in the visible range. For example, specific 
activator couples have characteristic emission spectra: ytterbium-erbium 
couples have emission maxima in the red or green portions of the visible 
spectrum, depending upon the phosphor host; ytterbium-holmium couples 
generally emit maximally in the green portion, ytterbium-thulium typically 
have an emission maximum in the blue range, and ytterbium-terbium usually 
emit maximally in the green range. For example, Y.sub.0.80 Yb.sub.0.19 
Er.sub.0.01 F.sub.2 emits maximally in the green portion of the spectrum. 
Although up-converting inorganic phosphor crystals of various formulae are 
suitable for use in the invention, the following formulae, provided for 
example and not to limit the invention, are generally suitable: 
Na(Y.sub.x Yb.sub.y Er.sub.z)F.sub.4 : x is 0.7 to 0.9, y is 0.09 to 0.29, 
and z is 0.05 to 0.01; 
Na(Y.sub.x Yb.sub.y Ho.sub.z)F.sub.4 : x is 0.7 to 0.9, y is 0.0995 to 
0.2995, and z is 0.0005 to 0.001; and 
Na(Y.sub.x Yb.sub.y Tm.sub.z)F.sub.4 : x is 0.7 to 0.9, y is 0.0995 to 
0.2995, and z is 0.0005 to 0.001. 
(Y.sub.x Yb.sub.y Er.sub.z)O.sub.2 S: x is 0.7 to 0.9, y is 0.05 to 0.12; z 
is 0.05 to 0.12. 
(Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.3 is a relatively 
efficient up-converting phosphor material. 
For exemplification, but not to limit the invention, 
ytterbium(Yb)-erbium(Er)-doped yttrium oxysulfides luminesce in the green 
after excitation at 950 nm. These are non-linear phosphors, in that the 
ytterbium acts as an "antenna" (absorber) for two 950 nm photons and 
transfers its energy to erbium which acts as an emitter (activator). The 
critical grain size of the phosphor is given by the quantum yield for 
green emission and the doping level of both Yb and Er, which is generally 
in the range of about 1 to 10 percent, more usually in the range of about 
2 to 5 percent. A typical Yb:Er phosphor crystal comprises about 10-30% Yb 
and about 1-2% Er. Thus, a phosphor grain containing several thousand 
formula units ensures the emission of at least one or more photons during 
a typical laser irradiation time. However, the nonlinear relationship 
between absorption and emission indicates that intense illumination at the 
excitation wavelength(s) may be necessary to obtain satisfactory signal in 
embodiments employing very small phosphor particles (i.e., less than about 
0.3 .mu.m). Additionally, it is usually desirable to increase the doping 
levels of activator/emitter couples for producing very small phosphor 
particles so as to maximize quantum conversion efficiency. 
Inorganic microcrystalline phosphors with rare earth activators generally 
have narrow absorption and line emission spectra. The line emission 
spectra are due to .function.-.function. transitions within the rare earth 
ion. These are shielded internal transitions which result in narrow line 
emission. 
In certain applications, such as where highly sensitive detection is 
required, intense illumination can be provided by commercially available 
sources, such as infrared laser sources (e.g., continuous wave (CW) or 
pulsed semiconductor laser diodes). For example, in applications where the 
microcrystalline phosphor particle must be very small and the quantum 
conversion efficiency is low, intense laser illumination can increase 
signal and decrease detection times. Alternatively, some applications of 
the invention may require phosphor compositions that have inherently low 
quantum conversion efficiencies (e.g., low doping levels of activator 
couple), but which have other desirable characteristics (e.g, 
manufacturing efficiency, ease of derivatization, etc.); such low 
efficiency up-converting phosphors are preferably excited with laser 
illumination at a frequency at or near (i.e., within about 25 to 75 nm) an 
absorption maximum of the material. The fact that no other light is 
generated in the system other than from the up-converting phosphor allows 
for extremely sensitive signal detection, particularly when intense laser 
illumination is used as the source of excitation radiation. Thus, the 
unique property of up-conversion of photon energy by up-converting 
phosphors makes possible the detection of very small particles of 
microcrystalline inorganic phosphors. For practical implementation of 
phosphors as ultrasensitive reporters, particularly as intracellular 
reporters, it is essential that the grain size of the phosphor be as small 
as practicable (typically less than about 0.3 to 0.1 .mu.m), for which 
laser-excited up-converting phosphors are well-suited. 
For example, various phosphor material compositions capable of 
up-conversion are suitable for use in the invention are shown in Table I. 
TABLE I 
______________________________________ 
Phosphor Material Compositions 
Host Material 
Absorber Ion 
Emitter Ion 
Color 
______________________________________ 
Oxysulfides (O.sub.2 S) 
Y.sub.2 O.sub.2 S 
Ytterbium Erbium Green 
Gd.sub.2 O.sub.2 S 
Ytterbium Erbium Red 
La.sub.2 O.sub.2 S 
Ytterbium Holmium Green 
Oxyhalides (OX.sub.y) 
YOF Ytterbium Thulium Blue 
Y.sub.3 OCl.sub.7 
Yterbium Terbium Green 
Fluorides (F.sub.x) 
YF.sub.3 Ytterbium Erbium Red 
GdF.sub.3 Ytterbium Erbium Green 
LaF.sub.3 Ytterbium Holmium Green 
NaYF.sub.3 Ytterbium Thulium Blue 
BaYF.sub.5 Ytterbium Thulium Blue 
BaY.sub.2 F.sub.8 
Ytterbium Terbium Green 
Gallates (Ga.sub.x O.sub.y) 
YGaO.sub.3 Ytterbium Erbium Red 
Y.sub.3 Ga.sub.5 O.sub.12 
Ytterbium Erbium Green 
Silicates (Si.sub.x O.sub.y) 
YSi.sub.2 O.sub.5 
Ytterbium Holmium Green 
YSi.sub.3 O.sub.7 
Ytterbium Thulium Blue 
______________________________________ 
In addition to the materials shown in Table I and variations thereof, 
aluminates, phosphates, and vanadates can be suitable phosphor host 
materials. In general, when silicates are used as a host material, the 
conversion efficiency is relatively low. In certain uses, hybrid 
up-converting phosphor crystals may be made (e.g., combining one or more 
host material and/or one or more absorber ion and/or one or more emitter 
ion). 
Exemplary up-converting phosphors excited at about 980 nm include, but are 
not limited to: Y.sub.0.80 Yb.sub.0.18 Er.sub.0.02)F.sub.3 ; Y.sub.0.87 
Yb.sub.0.13 Tm.sub.0.001)F.sub.3 ; Y.sub.0.80 Yb.sub.0.198 
Ho.sub.0.002)F.sub.3 ; Gd.sub.0.80 Yb.sub.0.18 Er.sub.0.02)F.sub.3 ; 
Gd.sub.0.87 Yb.sub.0.13 Tin.sub.0.001)F.sub.3 ; Gd.sub.0.80 Yb.sub.0.198 
Ho.sub.0.002)F.sub.3 ; Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.2 
S; Y.sub.0.87 Yb.sub.0.13 Tm.sub.0.001).sub.2 O.sub.2 S; Y.sub.0.80 
Yb.sub.0.198 Ho.sub.0.002).sub.2 O.sub.2 S; Gd.sub.0.86 Yb.sub.0.08 
Er.sub.0.06).sub.2 O.sub.2 S; Gd.sub.0.87 Yb.sub.0.13 Tin.sub.0.001).sub.2 
O.sub.2 S; Gd.sub.0.80 Yb.sub.0.198 Ho.sub.0.002).sub.2 O.sub.2 S; 
Exemplary up-converting phosphors excited at about 1500 nm include, but are 
not limited to: Y.sub.0.96 Er.sub.0.06).sub.2 O.sub.2 S; Gd.sub.0.96 
Er.sub.0.06).sub.2 O.sub.2 S. 
Preparation of Inorganic Phosphor Labels 
Techniques and methods for manufacture of inorganic phosphors has been 
described in the art. Up-converting phosphor crystals can be manufactured 
by those of ordinary skill in the art by various published methods, 
including but not limited to the following: Yocom et al. (1971) 
Metallurgical Transactions 2: 763; Kano et al. (1972) J. Electrochem. 
Soc., p. 1561; Wittke et al. (1972) J. Appl. Physics 43: 595; Van Uitert 
et al. (1969) Mat. Res. Bull. 4: 381; which are incorporated herein by 
reference. Other references which may be referred to are: Jouart JP and 
Mary G (1990) J. Luminescence 46: 39; McPherson GL and Meyerson SL (1991) 
Chem. Phys. Lett. (April) p.325; Oomen et al. (1990) J. Luminescence 46: 
353; NI H and Rand SC (1991) Optics Lett. 16 (Sept.); McFarlane RA (1991) 
Optics Lett. 16 (Sept.); Koch et al. (1990) Appl. Phys. Lett. 56: 1083; 
Silversmith et al. (1987) Appl. Phys. Lett. 51: 1977; Lenth W and 
McFarlane RM (1990) J. Luminescence 45: 346; Hirao et al. (1991) J. 
Non-crystalline Solids 135: 90; McFarlane et al. (1988) Appl. Phys. Lett. 
52: 1300, incorporated herein by reference). 
In general, inorganic phosphor particles are milled to a desired average 
particle size and distribution by conventional milling methods known in 
the art, including milling in a conventional barrel mill with zirconia 
and/or alumina balls for periods of up to about 48 hours or longer. 
Phosphor particles used in binding assays are typically about 3.0 to 0.01 
.mu.m in diameter (or along the long axis if nonspherical), more usually 
about 2.0 to 0.1 .mu.m in size, and more conveniently about 1.0 to 0.3 
.mu.m in size, although phosphor particles larger or smaller than these 
dimensions may be preferred for certain embodiments. Phosphor particle 
size is selected by the practitioner on the basis of the desired 
characteristics and in accordance with the guidelines provided herein. 
Fractions having a particular particle size range may be prepared by 
sedimentation, generally over an extended period (i.e., a day or more) 
with removal or the desired size range fraction after the appropriate 
sedimentation time. The sedimentation process may be monitored, such as 
with a Horiba Particle Analyzer. 
However, milling crystalline materials has several weaknesses. With 
milling, the particle morphology is not uniform, as milled particles 
result from random fracture of larger crystalline particles. Since the 
sensitivity of a detection assay using up-converting inorganic phosphors 
depends on the ability to distinguish between bound and unbound phosphor 
particles, it is preferable that the particles be of identical size and 
morphology. Size, weight, and morphology of up-converting microcrystalline 
phosphor particles can affect the number of potential binding sites per 
particle and thus the potential strength of particle binding to reporter 
and/or analyte. Monodisperse submicron spherical particles of uniform size 
can be generated by homogeneous precipitation reactions at high dilutions. 
For example, small yttrium hydroxy carbonate particles are formed by the 
hydrolysis of urea in a dilute yttrium solution. Similarly, up-converting 
inorganic phosphors can be prepared by homogeneous precipitation reactions 
in dilute conditions. For example, (Y.sub.0.86 Yb.sub.0.08 
Er.sub.0.06.sub.).sub.2 O.sub.3 was prepared as monodisperse spherical 
particles in the submicron size range by precipitation. 
However, after precipitation it is typically necessary to anneal the oxide 
in air at about 1500.degree. C., which can cause faceting of the spherical 
particles which can generate aggregate formation. Faceting can be 
substantially reduced by converting the small spherical particles of the 
oxide or hydroxy carbonate precursor to the oxysulfide phase by including 
a polysulfide flux for annealing. Using this technique, highly efficient 
oxysulfide particles in the 0.3 to 0.4 .mu.m diameter range were prepared 
as a dispersion in water. Frequently, sonication can be used to produce a 
monodisperse mixture of discrete spherical particles. After fractionation 
and coating, these particles can be used as up-converting reporters. 
Furthermore, this general preparative procedure is suitable for preparing 
much smaller phosphor particles (e.g., 0.1 .mu.m diameter or smaller), 
which may be advantageous for various assay formats. 
Frequently, such as with phosphors having an oxysulfide host material, the 
phosphor particles are preferably dispersed in a polar solvent, such as 
acetone or DMSO and the like, to generate a substantially monodisperse 
emulsion (e.g., for a stock solution). Aliquots of the monodisperse stock 
solution may be further diluted into an aqueous solution (e.g., a solution 
of avidin in buffered water or buffered saline). 
It was found that washing phosphors in acetone or DMSO improved 
suspendability of inorganic phosphor particles in water. In particular, 
the phosphor particles prepared with polysulfide flux are preferably 
resuspended and washed in hot DMSO and heated for about an hour in a steam 
bath then allowed to cool to room temperature under continuous agitation. 
The phosphor particles may be pre-washed with acetone (typically heated to 
boiling) prior to placing the particles in the DMSO. Hot DMSO-treated 
phosphors were found to be reasonably hydrophilic and form stable 
suspensions. A Microfluidizer.TM. (Microfluidics Corp.) can be used to 
further improve the dispersion of particles in the mixture. DMSO-phosphor 
suspensions can be easily mixed with water, preferably with small amounts 
of surfactant present. In general, polysaccharides (e.g., guar gum, 
xanthan gum, gum arabic, alginate, guaiac gum) can be used to promote 
deaggregation of particles. In a variation, particles are washed in hot 
DMSO and serially diluted into a 0.1% aqueous gum arabic solution, which 
appears to virtually eliminate water dispersion problems of phosphors. 
Resuspended phosphors in organic solvent, such as DMSO, are typically 
allowed to settle for a suitable period (e.g., about 1-3 days), and the 
supernatant which is typically turbid is used for subsequent conjugation. 
Ludox.TM. is a colloidal silica dispersion in water with a small amount of 
organic material (e.g., formaldehyde, glycols) and a small amount of 
alkali metal. Ludox.TM. and its equivalents can be used to coat 
up-converting phosphor particles which can subsequently be fired to form a 
ceramic silica coating which cannot be removed from the phosphor 
particles, but which can be readily silanized with organofunctional 
silanes (containing thiol, primary amine, and carboxylic acid 
functionalities) using standard silanization chemistries (Arkles, B, in: 
Silicon Compounds: Register and Review; 5th Edition (1991); Anderson, RG, 
Larson, GL, and Smith, C, eds.; p.59-64, Huls America, Piscataway, N.J.). 
Phosphor particles can be coated or treated with surface-active agents 
(e.g., anionic surfactants such as Aerosol OT) during the milling process 
or after milling is completed. For example, particles may be coated with a 
polycarboxylic acid (e.g., Additon XW 330, Hoechst, Frankfurt, Germany or 
Tamol, see Beverloo et al. (1992) op.cit.) during milling to produce a 
stable aqueous suspension of phosphor particles, typically at about pH 
6-8. The pH of an aqueous solution of phosphor particles can be adjusted 
by addition of a suitable buffer and titration with acid or base to the 
desired pH range. Depending upon the chemical nature of the coating, some 
minor loss in conversion efficiency of the phosphor may occur as a result 
of coating, however the power available in a laser excitation source can 
compensate for such reduction in conversion efficiency and ensure adequate 
phosphor emission. 
In general, preparation of inorganic phosphor particles and linkage to 
binding reagents is performed essentially as described in Beverloo et al. 
(1992) op.cit., and Tanke U.S. Pat. No. 5,043,265. Alternatively, a 
water-insoluble polyfunctional polymer which exhibits glass and melt 
transition temperatures well above room temperature can be used to coat 
the up-converting phosphors in a nonaqueous medium. For example, such 
polymer functionalities include: carboxylic acids (e.g., 5% acrylic 
acid/95% methyl acrylate copolymer), amine (e.g., 5% aminoethyl 
acrylate/95% methyl acrylate copolymer) reducible sulfonates (e.g., 5% 
sulfonated polystyrene), and aldehydes (e.g, polysaccharide copolymers). 
The phosphor particles are coated with water-insoluble polyfunctional 
polymers by coacervative encapsulation in nonaqueous media, washed, and 
transferred to a suitable aqueous buffer solution to conduct the 
heterobifunctional crosslinking to a protein (e.g., antibody) or 
polynucleotide probe molecule. An advantage of using water-insoluble 
polymers is that the polymer microcapsule will not migrate from the 
surface of the phosphor upon aging the encapsulated phosphors in an 
aqueous solution (i.e., improved reagent stability). Another advantage in 
using copolymers in which the encapsulating polymer is only partially 
functionalized is that one can control the degree of functionalization, 
and thus the number of biological probe molecules which can be attached to 
a phosphor particle, on average. Since the solubility and coacervative 
encapsulation process will depend on the dominant nonfunctionalized 
component of the copolymer, the functionalized copolymer ratio can be 
varied over a wide range to generate a range of potential crosslinking 
sites per phosphor, without having to substantially change the 
encapsulation process. 
A preferred functionalization method employs heterobifunctional 
crosslinkers that can be made to link the biological macromolecule probe 
to the insoluble phosphor particle in three steps: (1) bind the 
crosslinker to the polymer coating on the phosphor, (2) separate the 
unbound crosslinker from the coated phosphors, and (3) bind the biological 
macromolecule to the washed, linked polymer-coated phosphor. This method 
prevents undesirable crosslinking interactions between biological 
macromolecules and so reduces irreversible aggregation as described by 
Tanke et al. Examples of suitable heterobifunctional crosslinkers, polymer 
coating functionalities, and linkable biological macromolecules include, 
but are not limited to: 
______________________________________ 
Coating Heterobifunctional 
Biological 
Functionality 
Crosslinker Macromolecule 
______________________________________ 
carboxylate 
N-hydroxysuccimide 
Proteins (e.g., 
1-ethyl-3-(3-dimethylamino 
Ab, avidin) 
propyl)-carbodiimide (EDC) 
primary amine 
N-5-azido-2-nitrobenzoyl 
All having 1.degree. amine 
oxysuccimide (ANB-NOS) 
N-succinimidyl(4-iodoacetyl) 
aminobenzoate (SIAB) 
thiol (reduced 
N-succinimidyl(4-iodoacetyl) 
Proteins 
sulfonate) aminobenzoate (SIAB) 
______________________________________ 
Binding Assays 
Up-converting phosphors and up-converting organic dyes are used as 
reporters (i.e., detectable markers) to label binding reagents, either 
directly or indirectly, for use in binding assays to detect and quantitate 
the presence of analyte(s) in a sample. Binding reagents are labeled 
directly by attachment to up-converting reporters (e.g., surface 
adsorption, covalent linkage). Binding reagents which can be directly 
labeled include, but are not limited to: primary antibodies (i.e., which 
bind to a target analyte), secondary antibodies (i.e., which bind to a 
primary antibody or prosthetic group, such as biotin or digoxygenin), 
Staphlococcus aureus Protein A, polynucleotides, streptavidin, and 
receptor ligands. Binding reagents can also be indirectly labeled; thus, a 
primary antibody (e.g., a rabbit anti-erb-B antibody) can be indirectly 
labeled by noncovalent binding to a directly labeled second antibody 
(e.g., a goat anti-rabbit antibody linked to an up-converting inorganic 
phosphor). Quantitative detection of the analyte-probe complex may be 
conducted in conjunction with proper calibration of the assay for each 
probe employed. A probe is conveniently detected under saturating 
excitation conditions using, for example, a laser source or focused 
photodiode source for excitation illumination. 
Specific binding assays are commonly divided into homogeneous and 
heterogeneous assays. In a homogeneous assay, the signal emitted by the 
bound labeled probe is different from the signal emitted by the unbound 
labeled probe, hence the two can be distinguished without the need for a 
physical separation step. In heterogeneous assays, the signal emitted from 
the bound and unbound labeled probes is identical, hence the two must be 
physically separated in order to distinguish between them. The classical 
heterogeneous specific binding assay is the radioimmunoassay (RIA) (Yalow 
et al. (1978) Science 200: 1245, which is incorporated herein by 
reference). Other heterogeneous binding assays include the radioreceptor 
assay (Cuatrecasas et al. (1974) Ann. Rev. Biochem. 43: 109), the sandwich 
radioimmunoassay (U.S. Pat. No. 4,376,110, which is incorporated herein by 
reference), and the antibody/lectin sandwich assay (EP 0 166 623, which is 
incorporated herein by reference). Heterogeneous assays are usually 
preferred, and are generally more sensitive and reliable than homogeneous 
assays. 
Whether a tissue extract is made or a biological fluid sample is used, it 
is often desirable to dilute the sample in one or more diluents that do 
not substantially interfere with subsequent assay procedures. Generally, 
suitable diluents are aqueous solutions containing a buffer system (e.g., 
50 mM NaH.sub.2 PO.sub.4 or 5-100 mM Tris, pH4-pH10), non-interfering 
ionic species (5-500 mM KCl or NaCl, or sucrose), and optionally a 
nonionic detergent such as Tween. When the sample to be analyzed is 
affixed to a solid support, it is usually desirable to wash the sample and 
the solid support with diluent prior to contacting with probe. The sample, 
either straight or diluted, is then analyzed for the diagnostic analyte. 
In the general method of the invention, an analyte in a sample is detected 
and quantified by contacting the sample with a probe-label conjugate that 
specifically or preferentially binds to an analyte to form a bound 
complex, and then detecting the formation of bound complex, typically by 
measuring the presence of label present in the bound complexes. A 
probe-label conjugate can include a directly labeled analyte-binding 
reagent (e.g, a primary antibody linked to an up-converting phosphor) 
and/or an indirectly labeled analyte-binding reagent (e.g., a primary 
antibody that is detected by a labeled second antibody, or a biotinylated 
polynucleotide that is detected by labeled streptavidin). The bound 
complex(es) are typically isolated from unbound probe-label conjugate(s) 
prior to detection of label, usually by incorporating at least one washing 
step, so as to remove background signal attributable to label present in 
unbound probe-label conjugate(s). Hence, it is usually desirable to 
incubate probe-label conjugate(s) with the analyte sample under binding 
conditions for a suitable binding period. 
Binding conditions vary, depending upon the nature of the probe-label 
conjugate, target analyte, and specific assay method. Thus, binding 
conditions will usually differ if the probe is a polynucleotide used in an 
in situ hybridization, in a Northern or Southern blot, or in solution 
hybridization assay. Binding conditions will also be different if the 
probe is an antibody used in an in situ histochemical staining method or a 
Western blot (Towbin et al. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76: 
4350, incorporated herein by reference). In general, binding conditions 
are selected in accordance with the general binding methods known in the 
art. For example, but not for limitation, the following binding conditions 
are provided for general guidance: 
For antibody probes: 
10-200 mM Tris, pH 6-8; usually 100 mM Tris pH 7.5 
15-250 mM NaCl; usually 150 mM NaCl 
0.01-0.5 percent, by volume, Tween 20 
1 percent bovine serum albumin 
4.degree.-37.degree. C.; usually 4.degree. to 15.degree. C. 
For polynucleotide probes: 
3-10x SSC, pH 6-8; usually 5x SSC, pH 7.5 
0-50 percent deionized formamide 
1-10x Denhardt's solution 
0-1 percent sodium dodecyl sulfate 
10-200 .mu.g/ml sheared denatured salmon sperm DNA 
20.degree.-65.degree. C., usually 37.degree.-45.degree. C. for 
polynucleotide probes longer than 50 bp, usually 55.degree.-65.degree. C. 
for shorter oligonucleotide probes 
Additional examples of binding conditions for antibodies and 
polynucleotides are provided in several sources, including: Maniatis et 
al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring 
Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume 152, 
Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San 
Diego, Calif.; Young and Davis (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80: 
1194, which are incorporated herein by reference. When the probe is a 
receptor ligand, such as IL-2, .beta.-interferon, or other polypeptide 
hormones, cytokines, or lymphokines, suitable binding conditions generally 
are those described in the art for performing the respective 
receptor-ligand binding assay. 
Various examples of suitable binding conditions useful in immunoassays and 
immunohistochemistry are discussed, for example, in Harlow and Lane, 
Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988), 
which is incorporated herein by reference. In general, suitable binding 
conditions for immunological reactions include an aqueous binding buffer 
containing a salt (e.g., 5-500 mM NaCl or KCl), a buffer (e.g., Tris or 
phosphate buffer at pH 4-10), and optionally a nonionic detergent (e.g., 
Tween). In some embodiments, proteinase inhibitors or stabilizers may be 
included. The binding reactions are conducted for a suitable binding 
period, which, for antibody reactions, are typically at least about 1 to 5 
minutes, preferably at least about 30 minutes to several hours, although 
typically less than about 24 hours, more preferably less than about a few 
hours or less. Binding reactions (including washes) are typically carried 
out a temperature range of about 0.degree. C. to about 45.degree. C., 
preferably about 4.degree. C. to about 20.degree.-25.degree. C. 
Binding assays, which include in situ hybridization, in situ binding 
assays, and immunohistochemical staining, are usually performed by first 
incubating the sample with a blocking or prehybridization solution, 
followed by incubating the sample with probe under binding conditions for 
a suitable binding period, followed by washing or otherwise removing 
unbound probe, and finally by detecting the presence, quantity, and/or 
location of bound probe. The step of detecting bound probe can be 
accomplished by detecting label, if the probe is directly labeled, or by 
incubating the bound complex(es) with a second binding reagent (e.g., 
streptavidin) that is labeled and which binds to the probe, thus 
accomplishing indirect labeling of the probe. 
Up-converting labels are attached to probe(s) or second binding reagents 
that specifically or preferentially bind to probe(s) by any of the various 
methodologies discussed herein. Additionally, up-converting phosphor 
particles can be encapsulated in microspheres and coated with a probe 
(e.g., a specific antigen or antibody) for use as a labeled probe in an 
immunodiagnostic assay or nucleic acid hybridization assay to detect an 
analyte in a sample, such as the presence of an antibody, virus, or 
antigen in a blood serum sample, according to the method of Hari et al. 
(1990) Biotechniques 9: 342, which is incorporated herein by reference. 
Microencapsulation of phosphor can be accomplished in several ways known 
in the art, including coating the phosphor with a monomer solution and 
polymerizing the monomer to generate a polymer shell encasing the phosphor 
particle. Phosphor particles embedded in a polymer coating, such as a gel 
coating, can be functionalized (e.g., with amino groups) for covalent 
attachment to a binding component. 
Similarly, up-converting phosphor particles can be coated with probe 
directly, either by surface adsorption, by multiple hydrogen bonding, by 
electrostatic interaction, by van der Waals binding, or by covalent 
linkage to a functional group on a functionalized inorganic phosphor 
particle (e.g., a vitroceramic phosphor), for example, by linking an amino 
acid side-chain amine or carboxylate group of a probe protein to a 
carboxylate or amine group, respectively, on a functionalized phosphor 
particle. 
In certain embodiments, such as where stearic and/or charge interference of 
a bulky up-converting phosphor particle inhibits binding of the linked 
binding reagent to a target, it is desirable to incorporate a molecular 
spacer between the phosphor particle and the binding reagent. For example, 
a derivatized microencapsulated phosphor or vitroceramic phosphor may be 
conjugated to a heterobifunctional reagent having a --(CH.sub.2).sub.n -- 
spacer, where n is usually an integer from about 2 to about 50, between 
terminal functional groups. Similarly, phosphors may be directly 
derivatized with derivatizing agents (e.g., omega-functionalized silanes) 
having long intramolecular spacer chains, wherein a functional group 
reactive with a desired binding reagent is separated from the surface of 
the phosphor by a spacer of usually at least about 15 .ANG. (i.e., the 
equivalent of about 10 --CH.sub.2 -- straight-chain groups). In some 
embodiments, labels are attached by spacer arms of various lengths to 
reduce potential stearic hindrance. Multiple layers of spacer arms may 
also be used (e.g., multiple layers of streptavidin-biotin linkages). 
Multiple Analyte Detection 
Since up-converting phosphors can be differentiated on the basis of the 
excitation and/or emission wavelength spectra, up-converting phosphors can 
be used to detect and discriminate multiple analyte targets, such as, for 
example, cell surface antigens or soluble macromolecules. 
For example, streptavidin, avidin, or another linker macromolecule (e.g., 
antidigoxigenin antibody) are attached, respectively, to each of two 
different phosphors (for illustration, designated here as Phosphor#1 and 
Phosphor#2) which differ in their absorption and/or emission spectra so as 
to facilitate discrimination of the two phosphors based on absorption 
and/or emission wavelengths; e.g., one phosphor may emit in the blue and 
the other may emit in the green. For example and not limitation, 
Na(Y.sub.0.80 Yb.sub.0.18 Er.sub.0.02)F.sub.4 emits predominantly in the 
green, and Na(Y.sub.0.73 Yb.sub.0.27 Tm.sub.0.001)F.sub.4 emits 
predominantly in the blue, and thus these two phosphors may be 
discriminated on the basis of their phosphorescent emissions. 
Alternatively, two phosphors may produce essentially similar emission 
spectra but may have different excitation wavelengths which provide a 
basis for their discrimination in multiple analyte detection. A first 
binding component (e.g., an antibody) that binds specifically to a first 
analyte species (e.g., a lymphocyte CD4 antigen) and incorporates biotinyl 
moieties which may be bound by streptavidin-Phosphor#1 conjugates can be 
used to quantitatively detect the presence of a first analyte in a sample 
(e.g., a serum sample) by measuring phosphorescence of Phosphor#1 in 
analyte-binding component complexes. A second binding component (e.g., a 
probe polynucleotide) that binds specifically to a second analyte species 
(e.g., an HIV-1 sequence) and incorporates digoxygenin moieties (e.g., 
11-UTP-digoxygenin) which may be bound by antidigoxigenin-Phosphor#2 
conjugates can be used to quantitatively detect the presence of a second 
analyte in the sample by measuring phosphorescence of Phosphor#2 in 
analyte-binding component complexes. Thus, by simultaneously or 
contemporaneously detecting the presence of multiple phosphor reporters 
having differentiable signal characteristics, multiple analytes may be 
quantitatively detected in a single sample. 
Sandwich Binding Assays 
Up-converting phosphors labels can be used as reporters for sandwich 
binding assays (U.S. Pat. No. 4,376,110, which is incorporated herein by 
reference). For example, a magnetic bead, such as a superparamagnetic 
immunobead or functionalized magnetizable polymer particle (Polysciences, 
Inc., Warrington, Pa.), can serve as the solid substrate which has an 
immobilized first binding component (e.g., an antibody, a polynucleotide, 
or a lectin) that binds to a first epitope (i.e., a binding locus: an 
antigenic determinant, sugar moiety, chemical substituent, or nucleotide 
sequence) of an analyte. The analyte binds to the first binding component 
and also to a second binding component (e.g., an antibody, a lectin, or a 
polynucleotide) which binds to a second epitope of the analyte. Thus, the 
analyte bridges the two binding components to form a sandwich complex 
which is immobilized with respect to the solid substrate. The second 
binding component typically has an attached or incorporated label, such as 
a biotinyl group which can be bound to a streptavidin-coated up-converting 
phosphor. Alternatively, the second binding component can be linked 
directly to an up-converting phosphor, such as through a covalent linkage 
with a functionalized vitroceramic up-converting phosphor. 
The sandwich complex comprises the first binding component, an analyte, and 
the second binding component, which is labeled, either directly or 
indirectly, with an up-converting reporter. The sandwich complex is thus 
immobilized on the solid substrate, although the solid substrate itself 
may be mobile (e.g., a superparamagnetic bead circulating in a sample 
slurry). The presence and amount of analyte(s) can be quantitatively 
measured by detecting the presence of up-converting reporter in sandwich 
complexes. 
For example, a solid substrate may have a plurality of distinct species of 
first binding component (e.g., an array of different oligopeptides affixed 
to a solid support). One or more of the species of first binding component 
may bind to a particular analyte (e.g., a muscarinic receptor) in an 
analyte solution that is in contact with the solid support. Binding of the 
analyte to one or more of the first binding component species may then be 
detected with a second binding component (e.g., an anti-muscarinic 
receptor antibody) labeled with an up-converting phosphor (either directly 
or through a biotinylated secondary antibody). 
Solid substrates can be attached to a first binding component which can 
bind more than one distinct analyte (e.g., may be immunocrossreactive or 
polyspecific) and/or can be attached to multiple first binding component 
species which can bind multiple distinct analytes. Similarly, multiple 
second binding component species with binding specificities for particular 
analytes can be employed. When multiple second binding component species 
are employed, it is typically desirable to label each second binding 
component species with a unique up-converting label that can be 
distinguished on the basis of its absorption and/or emission properties. 
It is possible to use different absorbers in combination with various 
emitters to produce a collection of phosphors having several 
differentiable combinations of excitation and emission spectra. For 
example but not limitation, six differentiable phosphors may be generated 
from two absorbers and three emitters. A first absorber, A.sub.1, has an 
excitation wavelength of .lambda..sub.A1, a second absorber, A.sub.2 had 
an excitation wavelength of .lambda..sub.A2, a first emitter, E.sub.1, has 
an emission line at .lambda..sub.E1, a second emitter, E.sub.2 has an 
emission line at .lambda..sub.E2, and a third emitter, E.sub.3, has an 
emission line at .lambda..sub.E3, The six phosphors may be differentiated 
and the signal from each individually quantitated by illuminating the 
sample with an excitation wavelength .lambda.A1 and detecting separately 
the emitted radiation at .lambda..sub.E1, .lambda..sub.E2, and 
.lambda..sub.E3, and separately illuminating the sample with .lambda.A2 
and detecting separately the emitted radiation at .lambda..sub.E1, 
.lambda..sub.E2, and .lambda..sub.E3. Table II shows the various 
absorber:emitter combinations and their excitation and emission 
wavelengths. 
TABLE II 
______________________________________ 
Absorber: Emitter Combination 
Excitation .lambda. 
Emission .lambda. 
______________________________________ 
A1:E1 .lambda.A1 
.lambda.E1 
A1:E2 .lambda.A1 
.lambda.E2 
A1:E3 .lambda.A1 
.lambda.E3 
A2:E1 .lambda.A2 
.lambda.E1 
A2:E2 .lambda.A2 
.lambda.E2 
A2:E3 .lambda.A2 
.lambda.E3 
______________________________________ 
Of course, additional absorber:emitter combinations are possible to provide 
more than six differentiable phosphor labels. 
It is also possible to utilize solid substrates of different types which 
may be distinguished (e.g., by size, color, density, magnetic properties, 
shape, charge) so that a particular type of solid substrate is associated 
with a particular species of first binding component. 
For example and not limitation, the following three brief examples are 
provided to explicate further possible applications of multiple analyte 
sandwich assay methods. 
Substrate Differentiation 
The following example describes the use of distinguishable substrate types 
to detect the presence of specific immunoglobulin idiotypes in a sample 
(e.g., a blood serum sample taken from a patient) which can provide 
diagnostic information about the immune status of a patient (e.g., is a 
patient seroreactive with a particular antigen). 
Large superparamagnetic beads are conjugated to an immunogenic Herpesvirus 
Type II envelope glycoprotein, medium-sized superparamagnetic beads are 
conjugated to HIV gp120 glycoprotein, and small superparamagnetic beads 
are conjugated to an immunogenic cytomegalovirus envelope glycoprotein. A 
serum sample is taken from a patient and is incubated with a mixture of 
the superparamagnetic beads under binding conditions to permit specific 
binding of immunoglobulins in the sample with the three immobilized viral 
glycoprotein species. The superparamagnetic beads are separated from the 
sample to remove non-specifically bound immunoglobulin and incubated with 
up-converting phosphor particles coated with Staphylococcus aureus Protein 
A, which binds to IgG, under binding conditions. Superparamagnetic beads 
having specifically bound IgG are thus labeled with the phosphor-Protein A 
conjugate. Large, medium, and small superparamagnetic beads are then 
separately illuminated with phosphor excitation electromagnetic radiation 
and time-gated emitted phosphorescence is detected. Background 
attributable to non-specific binding, if any, is determined and subtracted 
using internal standard beads (bovine serum albumin coated 
superparamagnetic beads) and positive and negative control serum samples. 
The intensity of phosphorescence associated with the large, medium, and 
small beads provides a measure of the amount of antibodies in the sample 
which are reactive with the Herpesvirus Type II envelope glycoprotein, HIV 
gp120 glycoprotein, and cytomegalovirus envelope glycoprotein, 
respectively. This information can be used to determine whether an 
individual patient has been infected with the HIV-1, human CMV, and/or 
Herpes Simplex Type II viruses. 
Phosphor Differentiation 
The following example describes the use of differentiable up-converting 
phosphors to detect the presence and relative abundance of particular 
isoforms of human APP (amyloid precursor protein) in a serum or brain 
biopsy sample. Various isoforms of APP arise in the brain as a consequence 
of alternative exon usage and/or alternative proteolytic processing 
pathways. Thus, although all APP isoforms may share a common, hypothetical 
epitope (X), a particular APP isoform may have a unique epitope (Y), while 
another APP isoform has a unique epitope (Z). It is possible that the 
relative abundance of a particular APP isoform in a sample may be of 
predictive value or may be pathognomonic for Alzheimer's Disease. 
Superparamagnetic beads are conjugated to an antibody that binds 
specifically to a common APP epitope (X) shared by all isoforms. A 
specific antibody reactive with the unique Y epitope is labeled with 
Phosphor #1, which is excited by wavelength .lambda..sub.1 and emits in a 
wavelength spectrum centered in the blue. A specific antibody reactive 
with the unique Z epitope is labeled with Phosphor #2, which is excited by 
a wavelength .lambda..sub.2 and emits in a wavelength spectrum centered in 
the green. A sample containing APP isoforms is incubated with the 
superparamagnetic beads and labeled specific antibodies under binding 
conditions. The superparamagnetic beads are retrieved from the sample, 
either individually or in bulk. The beads are illuminated with wavelength 
.lambda..sub.1 and blue light emission is detected and measured, and 
illuminated with .lambda..sub.2 and green light emission is detected and 
measured. The intensity of .lambda..sub.1 -induced blue emission is a 
measure of the APP isoform(s) having the Y epitope, while the intensity of 
the .lambda..sub.2 -induced green emission is a measure of the APP 
isoform(s) having the Z epitope. If the emissions from two phosphors are 
readily distinguishable, .lambda..sub.1 and .lambda..sub.2 may be 
identical. The standardized relative intensities of the two phosphors 
provides a measure of the relative abundance of the APP isoform(s) 
containing the Y or Z epitopes. 
Phosphor and Substrate Differentiation 
The following example describes the use of differentiable up-converting 
phosphors in conjunction with distinguishable substrate types to detect 
the presence and relative abundance of particular T lymphocyte 
subpopulations in a blood sample taken from an individual. Although 
described here with reference to detecting T cell subpopulations, analyte 
multiplexing (i.e., detecting and/or characterizing multiple analytes in a 
sample by using various solid substrate types and/or up-converting 
phosphor labels) is believed to be a generally applicable method. 
Large superparamagnetic beads are conjugated to an anti-CD4 antibody, 
medium-sized superparamagnetic beads are conjugated to anti-CD8 antibody, 
and small superparamagnetic beads are conjugated to an anti-CD28 antibody. 
An antibody that specifically binds to the CD2 antigen is labeled with an 
up-converting phosphor that has an excitation wavelength .lambda..sub.1 
and emits in the red. An antibody that specifically binds to the CD45R 
antigen is labeled with an up-converting phosphor that has an excitation 
wavelength .lambda..sub.2 and emits in the green. An antibody that 
specifically binds to the CDw60 antigen is labeled with an up-converting 
phosphor that has an excitation wavelength .lambda..sub.3 and emits in the 
blue. 
A blood (or serum, sputum, urine, feces, biopsy tissue, etc.) sample is 
taken from a patient and is incubated with a mixture of the 
superparamagnetic beads and phosphor-labeled antibodies under binding 
conditions to permit specific binding of cells in the blood sample with 
the three bead-immobilized antibody species and the three phosphor-labeled 
antibody species. After antigen-antibody binding occurs, the 
superparamagnetic beads are segregated and examined, either sequentially 
or simultaneously, by illumination with .lambda..sub.1, .lambda..sub.2, 
and .lambda..sub.3, and quantitative detection of red, green, and blue 
emissions, respectively. For example, the intensity of .lambda..sub.1 
-induced red light emission associated with the large beads is a rough 
measure of the amount of cells having both CD4 and CD2 surface antigens 
and/or the relative abundance of those surface antigens (e.g., there may 
be very few CD4.sup.+ cells that have CD2, but those few cells may have a 
large amount of CD2 antigen, and hence a large CD2 phosphorescent signal). 
Similarly, the intensity of .lambda..sub.2 -induced green light associated 
with the large beads is a rough measure of the amount of cells having both 
CD4 and CD45R surface antigens and/or the relative abundance of those 
surface antigens in a sample. 
In this manner, an analyte sample, such as a blood sample, can be 
"fingerprinted" for the presence and relative distribution(s) (e.g., 
cosegregation and/or correlation) of various analyte species. Such an 
analyte fingerprint may be used for providing diagnostic or therapeutic 
information, for example, as to measuring a patient's immune status or 
measuring response to chemotherapy directed against a particular blood 
cell subset. Similar analyte fingerprints can be used to type pathogenic 
organisms and viruses, as well as to order polynucleotide sequences for 
gene mapping and/or sequencing. 
Superparamagnetic beads which can be differentiated based on size, shape, 
color, or density can be magnetically trapped individually and scanned 
with appropriate excitation illumination(s) and phosphor emission(s) 
characteristic of particular analytes detected. For example, a unitary 
detector can simultaneously or contemporaneously trap the 
superparamagnetic bead from a suspension, determine the bead type (size, 
shape, and/or color), and scan for presence and abundance of particular 
phosphors (by illuminating with excitation wavelength(s) and detecting 
emitted wavelengths). 
By performing binding assays under dilute conditions wherein an average of 
one analyte or less (e.g., lymphocyte) is bound per microbead, it is 
possible to type cells individually (e.g., determine the abundance of 
CD45R on each individual CD4.sup.+ cell) and thus generate more precise 
lymphocyte subpopulation definitions. 
Biotinylated magnetic beads can also be used to monitor the kinetics of 
binding streptavidin to phosphor particles and/or to segregate or purify 
streptavidin-coated up-converting phosphor particles from a reaction. 
Thus, streptavidin and up-converting phosphor particles are mixed in a 
reaction vessel under binding conditions for forming streptavidin-coated 
phosphor particles. After a suitable binding period, unbound streptavidin 
may be removed (e.g., by centrifugation wherein phosphor particles are 
collected as the pellet, unbound streptavidin in the supernatant is 
decanted, and the pellet is resuspended), biotinylated magnetic beads are 
added to the remaining phosphor suspension in binding conditions, and 
streptavidin-coated phosphor particles are recovered bound to the 
biotinylated magnetic beads. 
Photophysical Catalysis by Up-Converting Phosphors 
Other applications of the invention employ phosphors as a photophysical 
catalyst linked to a probe, where the radiation emitted by the phosphor is 
used, typically in conjunction with a dye molecule, to produce localized 
intense electromagnetic radiation in an area adjacent to the probe for 
various purposes other than detection (e.g., cytotoxicity, ionization of 
chemical species, mutagenesis, etc.). For example, an antibody that 
specifically binds to a cell surface antigen, such as a CD8 antigen on a 
CD8.sup.+ lymphocyte, may be used as a probe linked to a up-converting 
phosphor to localize the phosphor to CD8.sup.+ lymphocytes. A sample 
containing CD8.sup.+ lymphocytes can be incubated with the anti-CD8.sup.+ 
probe-phosphor conjugate and irradiated with an excitation wavelength 
(e.g., from an infrared laser diode), resulting in emission of upshifted 
photons (i.e., higher frequency electromagnetic radiation) in the vicinity 
of CD8.sup.+ lymphocytes to which the anti-CD8.sup.+ probe-phosphor 
conjugate has bound. The emitted radiation may be of a wavelength that is 
directly mutagenic and/or cytotoxic (e.g., ultraviolet radiation that can 
lead to formation of thymine dimers, 760-765 nm light is also believed to 
produce chromosomal damage) or may be of a wavelength that can cause a 
photolytic decomposition of a chemical present in the environment, leading 
to local formation of reactive species that may damage adjacent cells 
(e.g., photodecomposition of buckminsterfullerene, C.sub.60, to C.sub.58 
and C.sub.2, may produce free radicals that may cause lipid peroxidation 
of cell membranes). 
Since phosphor-emitted radiation is isotropic, it is generally desirable to 
physically separate targets (e.g., CD8.sup.+ lymphocytes) from 
non-targets (e.g., CD8.sup.- lymphocytes) prior to excitation irradiation, 
so that undesirable damage to non-targets by isotropic emission(s) (i.e., 
"secondary damage") is avoided. Physical separation may be accomplished by 
various means, including but not limited to: (1) performing excitation 
irradiation on a dilute suspension of target and non-target cells, wherein 
the mean distance separating individual cells is sufficient to reduce 
secondary damage to non-targets, and (2) employing hydrodynamic focusing 
to pass cells (both targets and non-targets) single file through an 
illumination zone (e.g., as in a fluorescence-activated cell sorter or the 
like). Thus, an up-converting phosphor linked to an anti-CD8.sup.+ 
antibody can be used to selectively damage CD8.sup.+ lymphocytes in a 
lymphocyte sample, where (1) the phosphor emits at a wavelength that is 
either directly cytotoxic and/or (2) the phosphor emits at a wavelength 
that produces reactive chemical species by photocatalysis of a compound 
present in the sample (e.g., a sample can be doped with 
buckminsterfullerene). 
Instead of using the emitted radiation directly for photocatalytic action 
on tissue or tumors, an excited form of oxygen, so called singlet excited 
oxygen (O.sub.2 '.DELTA.g) can be generated by energy transfer from a dye 
sensitizer to dissolved molecular oxygen. This scheme makes use of the 
tissue penetrating power of near-infrared radiation (red and ultrared 
region light, including 970 nm) which reaches the inorganic up-converting 
phosphor. Two of the infrared photons are converted either into a red, 
green, or blue photon depending on the absorption spectrum of the 
sensitizer dye. The dye is excited by the up-converted radiation into a 
triplet state which transfers its energy to a dissolved molecular oxygen 
molecule to yield an excited (singlet) oxygen molecule. The cytotoxic 
activity of singlet oxygen is well documented in photodynamic therapy and 
other biomedical applications (see, Wagnieres et al. (19-21 Jan. 1990) 
Future Directions and Applications of Photodynamic Therapy, pp. 249, SPIE 
Institutes for Advanced Optical Technologies, Society of Photo-Optical 
Instrumentation Engineers, Box 10, Bellingham, Wash. 98277; Pelegrin et 
al. (1991) Cancer 67: 2529; Wagnieres et al. (24-25 May 1991) Future 
Directions and Applications of Photodynamic Therapy, pp. 219; Folli et al. 
(17 Dec. 1991) Fluoresceine Clinique 4; Braichotte et al. (May 1991) 
ENT-Clinic, Lausanne, Switzerland). 
In this application the up-converting phosphor is mixed or laced with a 
sensitizing dye such as methylene blue, rose bengal or phthalocyanine 
derivatives, such as Zn-phthalocyanine. In the first and third case a 
red-emitting phosphor is used, whereas for rose bengal a green-emitting 
phosphor is best suited. The phthalocyanine derivatives are ideally suited 
for this purpose because of their total insolubility in aqueous or 
biological solutions. These dyes therefore stay in close proximity to the 
emitters so that the specificity of the cell surface-reporter/probe/dye 
complex becomes the limiting factor. In this case, specialized 
combinations of reporter/probe/dye formulations preferably in the 0.1 to 
0.3-micron size range must be synthesized in order to enable efficient 
energy transfer: first, up-converted radiation is absorbed by the dye as 
completely as possible; and second, the dye excited energy (triplet state) 
is transferred to dissolved molecular oxygen. Both processes are very 
efficient if the absorption spectrum of the sensitizer dye is matched to 
the up-converted radiation. 
This scheme presents a step beyond the traditional photodynamic therapy 
methods in that the red light can be used both for tracking and diagnostic 
as well as for therapeutic purposes after up-converting thus necessitating 
only one (infrared) light source at about 1000 nm. A further advantage is 
the greater range within biological samples of the infrared radiation 
compared to other known photodynamic therapy excitation schemes (750-850 
nm). 
For embodiments employing up-converting phosphors as photophysical 
catalysts, it is generally desirable that: (1) the wavelength(s) of the 
excitation radiation do not produce significant photocatalysis of the 
substrate compound, (2) the wavelength(s) of the excitation radiation are 
not directly cytotoxic or mutagenic, and (3) the emitted radiation is 
directly cytotoxic and/or is of an appropriate wavelength to produce a 
biologically effective amount of photodecomposition of a substrate 
compound (e.g., buckminsterfullerene, psoralen, compounds containing azide 
substituents or other photoactivated groups). Alternatively, histidine 
side chains of polypeptides can be oxidized by light in the presence of 
dye sensitizers, such as methylene blue or rose bengal (Proteins, 
Structures and Molecular Principles, (1984) Creighton (ed.), W. H. Freeman 
and Company, New York; Introduction to Protein Structure, (1991), C. 
Branden and J. Tooze, Garland Publishing, New York, N.Y., which are 
incorporated herein by reference). Thus, for example, up-converting 
phosphors linked to anti-CD8 antibodies can be used as photophysical 
catalysts to produce selective, localized damage to CD8.sup.+ lymphocytes. 
In accordance with the invention, essentially any antibody can be linked 
to an appropriate up-converting phosphor, either directly or by 
conjugation to protein A which may then bind the immunoglobulin. Thus, the 
up-converting photophysical catalysts of the invention may be used to 
target essentially any desired antigen or cell type that can be 
distinguished by the presence of an identified antigen. 
Up-converting Chelates 
Certain applications require small reporters. For example, the transport, 
ability to stay in suspension, the bonding dynamics, and the tendency 
toward removal by microphages may be improved for smaller reporters. 
However, the reduced sensitivity available with smaller reporters must 
also be considered. 
One type of small up-converting inorganic phosphor consists of rare earth 
ions in chelates. The use of lanthanide chelates as reporters has been 
developed for biological assays as described on pages 6 and 7 of this 
application. This prior use of lanthanide chelates involved 
down-conversion. That is, the emission light is at a wavelength which is 
longer than the excitation wavelength. 
Rare earth chelates may be used as up-converting reporters through stepwise 
excitation such as shown in FIG. 5a, or in FIG. 5b (except that all levels 
would be in the same ion). Energy transfer from a sensitizer ion to an 
activator ion cannot be used in the case of a single rare earth ion. 
Chelates suitable for use as up-converting phosphors include 
ethylenediaminetetraacetic acid (EDTA), dipicolinic acid (DPA), 
diethylenetriaminetetraacetic acid (DTTA), diethylenetriaminepentaacetic 
acid (DTPA), tetraazacyclotetradecanetetraacetic acid (TETA), as well as 
antibiotics, natural chelating proteins, phthalocyanines, and cryptates. 
Methods for preparation of lanthanide chelates and their use in biological 
assays are described in the literature (Mukkala et al. (1989) Anal. 
Biochem. 176: 319, Hemmila et al. (1984) Anal. Biochem. 137: 335, Soini 
and Kojola (1983) Clin. Chem. 29: 65, Nonisotopic DNA Probe Techniques 
(1992) Kricka (Ed.) Academic Press, New York, as well as the references on 
page 6 of this application). Up-conversion phosphor reporters can also 
consist of rare earth ions inside cage compounds such as fullerene 
materials following the procedures described by Bethune et al. (1993) 
Nature 366: 123 and references therein. 
Suitable ions for up-conversion in chelates include erbium, neodymium, 
thulium, holmium, and praseodymium. Other candidate ions include the other 
lanthanide elements, the actinide elements, and other metal elements. 
Stepwise excitation schemes suitable for up-conversion in lanthanide 
chelates are described in the literature on up-conversion lasers. Examples 
include up-conversion in erbium (Silversmith et al. (1986) J. Opt. Soc. 
Am. A3: 128, and Macfarlane et al. (1989) Appl. Phys. Lett. 54: 2301), 
neodymium (Macfarlane et al. (1988) Appl. Phys. Lett. 52: 1300), thulium 
(Nguyen et al. (1989) Appl. Opt. 28: 3553 and Allain et al. (1990a) 
Electron. Lett. 226: 166), holmium (Allain et al. (1990b) Electron. Lett. 
26: 261), and praseodymium (Smart et al. (1991) Electron. Lett. 27: 1307). 
Other up-conversion laser schemes that rely on energy transfer, energy 
pooling, cross relaxation, or avalanche absorption are not appropriate for 
up-converting chelates because they rely on energy transfer between ions. 
These processes are described by Auzel (1973) Proc. IEEE 61: 758 and Lenth 
and Macfarlane (March 1992) Optics and Photonic News 3: 8. Energy transfer 
can be efficient in a crystalline host containing many rare earth ions, 
but not in a solution where the concentration of ions is low and the 
phonon structure is less constrained. 
In certain cases, these schemes may not function as well for up-conversion 
in chelates. For example, certain of the schemes have been demonstrated 
using crystalline host materials at very low temperatures, and may not 
function as well at room temperature in a chelate. Schemes that do not 
involve intermediate relaxation such as that of Smart et al. have 
advantages in chelates because they can be excited more effectively with 
pulsed sources. Higher peak powers can be obtained from diode lasers when 
they are operated in a pulsed mode. The higher peak powers lead to more 
efficient up-conversion due to the nonlinear dependence on excitation 
power. 
Up-Converting Organic Dyes 
Similar to the up-converting inorganic phosphor reporters we propose to use 
"molecular" labels whose fluorescence will be detected by optoelectronic 
means. Infrared or red light is exciting the probe-reporter complex bound 
to a target, after which light is emitted at shorter wavelengths with 
respect to the illuminating source. This up-converted light is free of 
scattered light from the source or autofluorescence by virtue of its 
higher energy. Furthermore, autofluorescence is greatly reduced by virtue 
of the excitation in the infrared or red spectral range. The light source 
is a pump laser whose pump pulses are short in order to achieve high 
powers and low energy in order to enable nonlinear optical processes in 
the dye. The goal is to excite the second excited singlet state (S.sub.2) 
in a dye with a ps pulse from a tunable dye laser using two red or 
infrared photons. After pumping the S.sub.2 state the dye relaxes within a 
few ps to the fluorescing state (S.sub.1) which can be detected by 
optoelectronic means. The goal of reaching the S.sub.2 state using two 
photons enables one to take advantage of the increasing two-photon cross 
sections as one approaches the S.sub.2 state using two-photon absorption. 
The non-resonant two-photon absorption cross sections are on the order of 
10.sup.-49 to 10.sup.-50 cm.sup.4 s, whereas the cross sections 
corresponding to S.sub.2 absorption are larger by two to three orders of 
magnitude. A few specific examples will be mentioned: in general cyanines, 
xanthenes, rhodamines, acridines and oxazines are well suited for this 
purpose. Blue dyes can also be used, but the excitation wavelength will be 
in the red. Rhodamine can be excited at 650 to 700 nm using two photons, 
and fluorescence is expected around 555 nm. Many IR dyes such as IR-140, 
IR-132 and IR-125 can be excited at 1060 nm using two photons of the 
Nd:YAG fundamental, and fluorescence is expected in the 850 to 950 nm 
range. An example of a blue dye is BBQ excited at 480 nm to reach the 
S.sub.2 state at 240 nm, and fluorescence is expected at 390 nm. Many of 
these dyes are only slightly soluble in aqueous solution and are either 
polar in nature (cyanines) or have polar substituents. Depending on the 
nature of the probe, no or only minimal attachment chemistry needs to be 
undertaken because of the abundance of functional groups on the dye 
chromophore. Several companies sell entire lines of dyes: examples are 
KODAK, Exciton and Lambda Physik. The scientific foundations of two-photon 
laser excitation in organic dye molecules have been treated in a few 
experimental papers: A. Penzkofer and W. Leupacher, Optical and Quantum 
Electronics 19 (1987), 327-349; C. H. Chen and M. P. McCann, Optics 
Commun. 63 (1987), 335; J. P. Hermann and J. Ducuing, Optics Commun. 6 
(1972), 101; B. Foucault and J. P. Hermann, Optics Commun. 15 (1975), 412; 
Shichun Li and C. Y. She, Optica Acta 29 (1982), 281-287; D. J. Bradley, 
M. H. R. Hutchinson and H. Koetser, Proc. R. Soc. Lond. A 329 (1972), 
105-119. 
Resonant Multiphoton Ionization 
At very high laser intensities the up-converting organic dyes are induced 
to absorb an additional exciting photon in the field of focussed laser 
radiation. At those high laser intensities the fluorescence is suppressed 
in favor of absorption of an additional photon. This process usually 
brings the organic dye molecules above the ionization limit in solution 
and they stabilize by emitting an electron into the solvent shell. The 
result of this three-photon interaction is a molecular ion and an attached 
or solvated electron. When this charge separation is taking place in an 
electric field, the charges drift and generate a voltage that can be 
detected in an extremely sensitive manner. This amounts to the measurement 
of the transient conductivity in the solvent system and is usually more 
sensitive than light detection. The disadvantage of this method is that it 
necessitates electrodes that sense the moving charges. In that sense it is 
not as non-invasive a method as light detection. On the other hand it 
bypasses the conversion of light into a photoelectric signal which 
represents an enormous advantage. Every optical system has a restricted 
viewing angle that reduces efficiency, whereas photoionization "senses" 
always close to 100% of the charges generated. Effectively, the non-linear 
interaction of the laser field converts every excited organic dye molecule 
into an electric pulse at sufficiently high field intensities that can be 
routinely achieved using commercial laser sources. Specific examples are 
the excitation of Rhodamine around 650 to 700 nm, or BBQ excitation around 
480 nm. Organic dyes absorbing in the red have to absorb two additional 
photons after being excited into S.sub.2 thus making the whole process a 
four-photon excitation process, which is slower than a three-photon 
non-linear process. There may, however, be circumstances where such a 
four-photon process is desirable. 
Detection Apparatus 
Detection and quantitation of inorganic up-converting phosphor(s) is 
generally accomplished by: (1) illuminating a sample suspected of 
containing up-converting phosphors with electromagnetic radiation at an 
excitation wavelength, and (2) detecting phosphorescent radiation at one 
or more emission wavelength band(s). 
Illumination of the sample is produced by exposing the sample to 
electromagnetic radiation produced by at least one excitation source. 
Various excitation sources may be used, including infrared laser diodes 
and incandescent filaments, as well as other suitable sources. Optical 
filters which have high transmissibility in the excitation wavelength 
range(s) and low transmissibility in one or more undesirable wavelength 
band(s) can be employed to filter out undesirable wavelengths from the 
source illumination. Undesirable wavelength ranges generally include those 
wavelengths that produce detectable sample autofluoresence and/or are 
within about 25-100 nm of excitation maxima wavelengths and thus are 
potential sources of background noise from scattered excitation 
illumination. Excitation illumination may also be multiplexed and/or 
collimated; for example, beams of various discrete frequencies from 
multiple coherent sources (e.g., lasers) can be collimated and multiplexed 
using an array of dichroic mirrors. In this way, samples containing 
multiple phosphor species having different excitation wavelength bands can 
be illuminated at their excitation frequencies simultaneously. 
Illumination may be continuous or pulsed, or may combine continuous wave 
(CW) and pulsed illumination where multiple illumination beams are 
multiplexed (e.g., a pulsed beam is multiplexed with a CW beam), 
permitting signal discrimination between phosphorescence induced by the CW 
source and phosphorescence induced by the pulsed source, thus allowing the 
discrimination of multiple phosphor species having similar emission 
spectra but different excitation spectra. For example but not limitation, 
commercially available gallium arsenide laser diodes can be used as an 
illumination source for providing near-infrared light. 
The ability to use infrared excitation for stimulating up-converting 
phosphors provides several advantages. First, inexpensive IR and near-IR 
diode lasers can be used for sustained high-intensity excitation 
illumination, particularly in IR wavelength bands which are not absorbed 
by water. This level of high-intensity illumination would not be suitable 
for use with conventional labels, such as ordinary fluorescent dyes (e.g., 
FITC), since high-intensity UV or visible radiation produces extensive 
photobleaching of the label and, potentially, damage to the sample. The 
ability to use higher illumination intensities without photobleaching or 
sample damage translates into larger potential signals, and hence more 
sensitive assays. 
The compatibility of up-converting labels with the use of diode lasers as 
illumination sources provide other distinct advantages over lamp sources 
and most other laser sources. First, diode laser intensity can be 
modulated directly through modulation of the drive current. This allows 
modulation of the light for time-gated or phase-sensitive detection 
techniques, which afford sensitivity enhancement without the use of an 
additional modulator. Modulators require high-voltage circuitry and 
expensive crystals, adding both cost and additional size to apparatus. The 
laser diode or light-emitting diode may be pulsed through direct current 
modulation. Second, laser illumination sources provide illumination that 
is exceptionally monochromatic and can be tightly focused on very small 
spot sizes, which provides advantages in signal-to-noise ratio and 
sensitivity due to reduced background light outside of the desired 
excitation spectral region and illuminated volume. A diode laser affords 
these significant advantages without the additional expense and size of 
other conventional or laser sources. 
Detection and quantitation of phosphorescent radiation from excited 
up-converting phosphors can be accomplished by a variety of means. Various 
means of detecting phosphorescent emission(s) can be employed, including 
but not limited to: photomultiplier devices, avalanche photodiode, 
charge-coupled devices (CCD), CID devices, photographic film emulsion, 
photochemical reactions yielding detectable products, and visual 
observation (e.g., fluorescent light microscopy). If the reporters are 
organic dyes, resonant multiphoton ionization can be sensed using 
electrostatic position-sensitive detectors. Detection can employ 
time-gated and/or frequency-gated light collection for rejection of 
residual background noise. Time-gated detection is generally desirable, as 
it provides a method for recording long-lived emission(s) after 
termination of illumination; thus, signal(s) attributable to 
phosphorescence or delayed fluorescence of up-converting phosphor is 
recorded, while short-lived autofluoresence and scattered illumination 
light, if any, is rejected. Time-gated detection can be produced either by 
specified periodic mechanical blocking by a rotating blade (i.e., 
mechanical chopper) or through electronic means wherein prompt signals 
(i.e., occurring within about 0.1 to 0.3 .mu.s of termination of 
illumination) are rejected (e.g., an electronic-controlled, solid-state 
optical shutter such as Pockel's or Kerr cells). Up-converting phosphors 
and up-converting delayed fluorescent dyes typically have emission 
lifetimes of approximately a few milliseconds (perhaps as much as 10 ms, 
but typically on the order of 1 ms), whereas background noise usually 
decays within about 100 ns. Therefore, when using a pulsed excitation 
source, it is generally desirable to use time-gated detection to reject 
prompt signals. 
Since up-converting phosphors are not subject to photobleaching, very weak 
emitted phosphor signals can be collected and integrated over very long 
detection times (continuous illumination or multiple pulsed illumination) 
to increase sensitivity of detection. Such time integration can be 
electronic or chemical (e.g., photographic film). When non-infrared 
photographic film is used as a means for detecting weak emitted signals, 
up-converting reporters provide the advantage as compared to 
down-converting phosphors that the excitation source(s) typically provide 
illumination in a wavelength range (e.g., infrared and near infrared) that 
does not produce significant exposure of the film (i.e., is similar to a 
darkroom safelight). Thus, up-converting phosphors can be used as 
convenient ultrasensitive labels for immunohistochemical staining and/or 
in situ hybridization in conjunction with fluorescence microscopy using an 
infrared source (e.g., a infrared laser diode) and photographic film 
(e.g., Kodak Ektachrome) for signal and image detection of visible range 
luminescence (with or without an infrared-blocking filter). 
Instrumentation Overview 
The basic purpose of the instrumentation is to expose the up-converting 
phosphor particles of an assay sample to near-infrared (NIR) light and to 
measure the amount of visible light that is emitted. 
FIG. 1 is an optical and electronic block diagram illustrating 
representative apparatus 10 for performing diagnostics on a sample 15 
according to the present invention. The invention may be carried out with 
one or a plurality of reporters. For purposes of illustration, the 
apparatus shows a system wherein two diagnostics are performed on a single 
sample in which two phosphor reporters are used. The first reporter has an 
excitation band centered at .lambda..sub.1 and an emission band centered 
at .lambda..sub.1 ' while the second reporter has respective excitation 
and emission bands centered at .lambda..sub.2 and .lambda..sub.2'. Since 
the reporters of the present invention rely on multiphoton excitation, 
wavelengths .lambda..sub.1 and .lambda..sub.2 are longer than wavelengths 
.lambda..sub.1 ' and .lambda..sub.2 '. The former are typically in the 
near infrared and the latter in the visible. 
A pair of light sources 20(1) and 20(2), which may be laser diodes or 
light-emitting diodes (LEDs), provide light at the desired excitation 
wavelengths, while respective detectors 22(1) and 22(2), which may be 
photodiodes, detect light at the desired emission wavelengths. The emitted 
radiation is related to the incident flux by a power law, so efficiency 
can be maximized by having the incident beam sharply focused on the 
sample. To this end, light from the two sources is combined to a single 
path by a suitable combination element 25, is focused to a small region by 
a lens or other focusing mechanism 27, and encounters the sample. Light 
emitted by the phosphor reporters is collected by a lens 30, and 
components in the two emission bands are separated by a suitable 
separation element 32 and directed to the respective detectors. 
There are a number of possible regimes for driving the laser diodes and 
detecting the emitted light in the different wavelength bands. This is 
shown generically as a control electronics block 35 communicating with the 
laser diodes and detectors. The particular timing and other 
characteristics of the control electronics will be described below in 
connection with specific embodiments. 
There may be a plurality of reporters having distinct emission bands but a 
common excitation band. In such a case, the system would include multiple 
detectors for a single laser diode. Similarly, there may be a plurality of 
reporters having distinct excitation bands but a common emission band. In 
such a case, the system would include multiple laser diodes for a single 
detector, and would use time multiplexing techniques or the like to 
separate the wavelengths. 
Light from the two sources is shown as being combined so as to be focused 
at a single location by a common focusing mechanism. This is not 
necessary, even if it is desired to illuminate the same region of the 
sample. Similarly, the collection need not be via a single collection 
mechanism. If it is necessary to preserve all the light, the combination 
and separation elements can include a wavelength division multiplexer and 
a demultiplexer using dichroic filters. If loss can be tolerated, 50% beam 
splitters and filters can be used. 
The schematic shows the light passing through the sample and being detected 
in line. As a general matter, the emission from the phosphor reporters is 
generally isotropic, and it may be preferred to collect light at an angle 
from the direction of the incident light to avoid background from the 
excitation source. However, since the excitation and the emission bands 
are widely separated, such background is unlikely to be an issue in most 
cases. Rather, other considerations may dictate other geometries. For 
example, it may be desired to detect light traveling back along the path 
of the incident radiation so that certain elements in the optical train 
are shared between the excitation and the detection paths. 
A typical type of instrument with shared elements is a microscope where the 
objective is used to focus the excitation radiation on the sample and 
collect the emitted radiation. A potentially advantageous variation on 
such a configuration makes use of the phenomenon of optical trapping. In a 
situation where the reporter is bound to a small bead, it may be possible 
to trap the bead in the region near the beam focus. The same source, or a 
different source, can be used to excite the reporter. The use of an 
infrared diode laser to trap small particles is described in Sato et al., 
"Optical trapping of small particles using a 1.3 .mu.m compact InGaAsP 
laser," Optics Letters, Vol. 16, No. 5 (Mar. 1, 1991), incorporated herein 
by reference. 
Specific Detection Techniques 
As outlined above, multichannel detection uses optical devices such as 
filters or dichroic beam splitters where the emission bands of the 
phosphor reporters are sufficiently separated. Similarly, it was pointed 
out that multiple reporters having a common emission band could be 
detected using electronic techniques. These electronic techniques will be 
described below in connection with multiple sources. However, the 
techniques will be first described in the context of a single channel. The 
techniques are useful in this context since there are sources of 
background that are in the same wavelength range as the signal sought to 
be measured. 
FIG. 2A shows an apparatus for implementing phase sensitive detection in 
the context of a single channel. Corresponding reference numerals are used 
for elements corresponding to those in earlier described figures. In this 
context, control electronics 35 comprises a waveform generator 37 and a 
frequency mixer 40. Waveform generator 37 drives laser diode 20(1) at a 
frequency f.sub.1, and provides a signal at f.sub.1 to the frequency 
mixer. The frequency mixer also receives the signal from detector 22(1) 
and a phase control input signal. This circuitry provides additional 
background discrimination because the background has a much shorter 
lifetime than the signal sought to be measured (nanoseconds or 
microseconds compared to milliseconds). This causes the signal and 
background to have different phases (although they are both modulated at 
the characteristic frequency of the waveform generator). For a discussion 
of the lifetime-dependent phase shift, see Demtroder, Laser Spectroscopy, 
Springer-Verlag, New York, 1988, pp. 557-559, incorporated herein by 
reference). The phase input signal is controlled to maximize the signal 
and discriminate against the background. This background discrimination 
differs from that typical for phase sensitive detection where the signal 
is modulated and the background is not. Discrimination against unmodulated 
background is also beneficial here, leading to two types of 
discrimination. 
Because the signal relies on two-photon excitation, it is possible to use 
two modulated laser diodes and to detect the signal at the sum or 
difference of the modulation frequencies. FIG. 2B shows such an 
arrangement where first and second laser diodes 20(1) and 20(1)' (emitting 
at the same wavelength .lambda..sub.1, or possibly different wavelengths) 
are modulated by signals from waveform generators 37a and 37b operating at 
respective frequencies f.sub.a and f.sub.b. The waveform generator output 
signals are communicated to a first frequency mixer 42, and a signal at 
f.sub.a .+-.f.sub.b is communicated to a second frequency mixer 45. The 
signal from detector 22(1) and a phase input signal are also communicated 
to frequency mixer 45. 
FIG. 3 shows apparatus for performing gated detection. Since the background 
is shorter-lived than the signal, delaying the detection allows improved 
discrimination. To this end, the laser diode is driven by a pulse 
generator 50, a delayed output of which is used to enable a gated 
integrator or other gated analyzer 55. 
FIG. 4 shows an apparatus for performing diagnostics on a sample using 
first and second, reporters having excitation bands centered at 
.lambda..sub.1 and .lambda..sub.2, and having overlapping emission bands 
near .lambda..sub.3. The sample is irradiated by light from laser diodes 
20(1) and 20(2) as discussed above in connection with FIG. 1. First and 
second waveform generators 37(1) and 37(2) drive the laser diodes at 
respective frequencies f.sub.1 and f.sub.2, and further provide signals at 
f.sub.1 and f.sub.2 to respective frequency mixers 60(1) and 60(2). The 
signal from detector 22(3) is communicated to both frequency mixers, which 
also receive respective phase input signals. Thus, frequency mixer 60(1) 
provides an output signal corresponding to the amount of emitted light 
modulated at frequency f.sub.1, which provides a measure of the presence 
of the first reporter in the sample. Similarly, frequency mixer 60(2) 
provides an output signal corresponding to the amount of emitted light 
modulated at frequency f.sub.2, which provides a measure of the presence 
of the second reporter in the sample. 
The use of two different wavelengths was discussed above in the context of 
two reporters having different excitation bands. However, the discussion 
is germane to a single reporter situation as well. Since the excitation is 
a two-photon process, there is no requirement that the two photons have 
the same energy. Rather, it is only necessary that the total energy of the 
two photons fall within the excitation band. Thus, since it is relatively 
straightforward and inexpensive to provide different wavelengths with 
laser diodes, there are more possible combinations, i.e., more possible 
choices of total excitation energy. This allows more latitude in the 
choice of rare earth ions for up-converters since the excitation steps 
need not rely on energy transfer coincidences involving a single photon 
energy. Further, it may be possible to achieve direct stepwise excitation 
of the emitting ion (the erbium ion in the example outlined above) without 
using energy transfer from another absorbing ion (the ytterbium ion in the 
example) while taking advantage of resonant enhancement of intermediate 
levels. Additionally, the use of different wavelengths for a single 
reporter can provide additional options for excitation-dependent 
multiplexing and background discrimination techniques. 
Multiple wavelength excitation of a single phosphor may occur in a number 
of ways, as shown in FIGS. 5A through 5C. Two lasers may cause stepwise 
excitation of a single ion, as shown in FIG. 5A. A first laser stimulates 
excitation from level 1 to level 2, and a second laser stimulates 
excitation from level 2 to level 3, at which level emission occurs. Single 
ion excitation can also occur using energy transfer as shown in FIG. 5B. 
In this case, a first laser stimulates excitation from level 1 to level 2, 
energy transfer occurs from level 2 to level 3, and a second laser 
stimulates excitation from level 3 to level 4. In a variation of the 
latter process, levels 1 and 2 can be in a first ion (i.e., a sensitizer 
ion) and levels 3 and 4 in a second ion (i.e., activator ion) as shown in 
FIG. 5C. 
In a stepwise excitation scheme shown in FIG. 5A, energy transfer is not 
required, and thus information on the polarization of the excitation 
lasers may be preserved and cause polarization of the emitted radiation. 
In this case, depolarization of the light may allow for enhanced 
discrimination between signal and background noise. 
For the multi-ion multi-laser excitation scheme shown in FIG. 5C, there may 
be several phosphors that share a common excitation wavelength. In this 
case, discrimination between different phosphors may be performed on the 
basis of different emission wavelengths and/or through time-gated, 
frequency-modulated, and/or phase-sensitive detection utilizing modulation 
of the excitation wavelength(s). 
Specific Instrument Embodiments 
FIG. 6 is a schematic view showing the optical train of a particular 
embodiment of apparatus for carrying out the present invention on a sample 
using a hand-held probe. This embodiment takes the form of a miniaturized 
instrument comprising a housing 75 (shown in phantom), a hand-held probe 
80, with a fiber optic connecting cable 82. The optical and electronics 
components are located within the housing. For purposes of illustration, 
the optical components of a 3-channel system are shown. The sample may 
contain up to three reporters having distinct emission bands, for example, 
in the blue, green, and red portions of the visible spectrum. It is also 
assumed that the reporters have distinct excitation bands in the near 
infrared. 
The output beams from three laser diodes 85a-c are communicated through 
graded index (GRIN) lenses 87a-c, focused onto the ends of respective 
fiber segments 88a-c and coupled into a single fiber 90 by a directional 
coupler 92 or other suitable device. The light emerging from the end of 
fiber 90 is collimated by a GRIN lens 95, passes through a dichroic beam 
splitter 97, and is refocused by a GRIN lens 100 onto the end of fiber 
optic cable 82. The beam splitter is assumed to pass the infrared 
radiation from the laser diodes but reflect visible light. 
Hand-held probe 80 includes a handpiece 102, an internal GRIN lens 105, and 
a frustoconical alignment tip 110. The light emerging from fiber 82 is 
focused by GRIN lens 105 at a focus point 115 that is slightly beyond 
alignment tip 110. The alignment tip is brought into proximity with the 
test tube holding the sample so that focus point 115 is in the sample. It 
is assumed that the test tube is transmissive to the laser radiation. 
A portion of the light emanating from the region of focus point 115 in the 
sample is collected by GRIN lens 105, focused into fiber 82, collimated by 
GRIN lens 100, and reflected at dichroic beam splitter 97. This light may 
contain wavelengths in up to the three emission bands. Optical filters 
120a-c direct the particular components to respective photodetectors 
125a-c. A particular filter arrangement is shown where each filter 
reflects light in a respective emission band, but other arrangements would 
be used if, for example, one or more of the filters were bandpass filters 
for the emission bands. 
The control electronics are not shown, but could incorporate the 
time-multiplexed or heterodyne techniques discussed above. Such techniques 
would be necessary, for example, if the emission bands were not distinct. 
FIG. 7A is a schematic of an embodiment of the invention in which a charge 
coupled device (CCD) imaging array 150 is used as a detector in 
combination with a two dimensional array 152 of peptides or other 
biologically active species deposited on a glass or plastic substrate. The 
CCD array has a number of individually addressable photosensitive detector 
elements 155 with an overlying passivation layer 157 while the peptide 
array has a number of individual binding sites 160. The probe containing 
the phosphor would be reaction specific to one or more of the elements in 
this peptide array and would therefore become physically attached to those 
elements and only those elements. The peptide array is shown as having a 
one-to-one geometric relation to the imaging array in which one pixel 
corresponds to each element in the peptide array. However, it is also 
possible to have larger peptide elements that cover a group of detector 
elements should such be necessary. 
Various of the techniques described above can be used to enable the 
detector array to distinguish the emissions of the phosphor from the 
infrared laser stimulation. 
First, it is possible to use a phosphor that responds to IR stimulation 
beyond the sensitivity range of the detector array. An example of such a 
phosphor would be Gadolinium oxysulfide: 10% Erbium. This phosphor is 
stimulated by 1.5-micron radiation and emits at 960 nm and 520 nm. The 
detector array is insensitive to 1.5-micron radiation but is sensitive to 
the up-converted radiation. 
Further, since the phosphor emission is relatively slow in rise and fall 
time it could be time resolved from a pulsed laser stimulation source by 
the CCD detector array. The decay time for the upconversion process is a 
variable dependent on the particular emitting transition and the phosphor 
host; however, it is normally in the range 500 .mu.s seconds to 10 ms. 
This is very slow compared to the laser excitation pulse and the 
capability of the detector array. 
The techniques for fabricating the CCD array are well-known since CCD 
imaging arrays have been commercially available for many years. A variety 
of such devices can be obtained from David Sarnoff Research Center, 
Princeton, N.J. 
The techniques for fabricating the peptide array are described in a paper 
by Fodor et al., "Light-Directed, Spatially Addressable Parallel Chemical 
Synthesis," Science, Vol. 251, pp. 767-773 (Feb. 15, 1991), incorporated 
herein by reference. The particular array described contains 1024 discrete 
elements in a 1.28 cm.times.1.28 cm area. 
The embodiment of FIG. 7A shows the peptide array in intimate contact with 
the CCD array.. Indeed it may be possible to deposit the peptides directly 
on the passivation layer without a separate substrate. However, there may 
be situations where spatially separated arrays are preferred FIG. 7B shows 
an embodiment where the peptide array and the CCD array are separated. An 
array of lenses 165 collect the light from respective binding sites and 
focus it on respective detector elements. This arrangement facilitates the 
use of filters to the extent that other techniques for rejecting the 
excitation radiation are not used. 
Optical trapping may be used to transiently immobilize a sample particle 
for determination of the presence or absence of phosphor on the particle. 
Conveniently, the wavelength range used to trap sample particles may be 
essentially identical to an excitation wavelength range for the 
up-converting phosphor(s) selected, so that optical trapping and 
excitation illumination is performed with the same source. FIG. 8 shows a 
block diagram of an apparatus used for single-beam gradient force trapping 
of small particles. 
FIG. 26 is a block diagram of one embodiment of apparatus for carrying out 
the present invention on a sample using a microscope. In this embodiment a 
standard microscope is modified to accept infrared scanning optics and 
image processing electronics. A suitable microscope for modification is 
the Zeiss model CLSM-10. 
The microscope is fitted with a HeNe laser A1 for visible imaging and an 
argon laser A2 for both visible and UV imaging. Both lasers are mounted 
internally and are individually selectable through a series of motorized 
shutters A3. The upconverting phosphors are excited with an externally 
mounted IR laser diode. In the preferred embodiment, two IR laser diodes 
A4 and A5, operating at two different IR wavelengths, are coupled to the 
microscope thereby allowing multiple phosphor reporters to be identified. 
Laser diodes A4 and A5 are individually selectable using motorized 
shutters A6. When an IR beam is selected, it is routed through the 
microscope's galvanometrically controlled scanning mirrors A7 which scan 
the beam in a raster fashion. The beam passes through the objective lens 
(not shown) onto a sample A8 and is reflected back through the objective 
lens to a set of galvanometrically controlled receiving mirrors A7. 
Receiving mirrors A7 reflect the light onto pinhole optics A9. If the 
confocal mode is selected, pinhole A9 limits the detected image to the 
light collected from the focal plane. The light is imaged on a 
photomultiplier tube (PMT) A10. The thickness of the focal plane is 
proportional to the size of the pinhole. The scanning speed is chosen such 
that sufficient signal intensity is received at the PMT A10. In the 
preferred embodiment of this apparatus, a 20 micrometer diameter pinhole 
is used which results in a depth of field of about 1 micrometer. If the 
confocal mode is not selected, the beam is deflected around pinhole optics 
A9 directly to PMT A10. 
Once the optical signal is converted into an electronic one, a standard, 
composite video signal can be developed and displayed as an image on a 
television monitor A11. The image can be manipulated and enhanced through 
standard image processing software. In the preferred embodiment of this 
apparatus the software runs on an IBM 486 PC A12. The software can be used 
to perform averaging, filtering, edge detection and overlaying the images 
received from each of the different light sources. 
In the confocal mode, it is possible to reconstruct a 3 dimensional view of 
sample A8. The reconstruction is formed by stepping through sample A8 at 
small intervals, making an image of the sample at each interval. The 
multiple sequential images are transferred to an external graphics machine 
(not shown) for reconstruction of the sample in 3 dimensions. These 3-D 
images can then be rotated to give different perspectives of the data 
sets, leading to a better understanding of the samples. 
FIG. 27 is a block diagram of a microtiter plate reader for use with the 
present invention. Within a light-tight test chamber B1 is a near IR laser 
excitation source B2, a photomultiplier tube (PMT) detector B3, and a 
sample assay plate B4. In the preferred embodiment of this apparatus, 
assay plate B4 is a Terasaki HLA plate. This plate is preferred due to its 
small tapered sample wells which tend to concentrate the sample material 
into a relatively small target area. The target area in this configuration 
is still larger than the diameter of the laser beam. Furthermore, it is 
possible that the distribution of the assay material across the bottom of 
the well is not even. Because of these two factors, simply aiming the 
laser at the center of the bottom well surface is unlikely to provide 
accurate readings. There are several approaches that can be used to 
circumvent this problem. The first approach is to defocus the laser beam 
sufficiently to allow a larger amount of the target area to be 
interrogated. However, depending upon the output of laser B2, defocussing 
the beam may lower the sensitivity of the apparatus to an unacceptable 
level. Another approach is to raster scan the laser beam across the bottom 
of target well. A third and preferred approach is to simply automate the 
scanning and data collection system. 
Light from laser B2 passes through a filter B5 and is focussed by a lens B6 
onto an individual sample well of assay plate B4. Plate B4 is mounted on a 
pair of translators B7 which allow positioning in the horizontal and 
vertical directions. In the present configuration translators B7 allow 
approximately 2.5 centimeters of travel; sufficient to address 3 sample 
wells in each direction. Translators B7 are controlled by an x-y 
controller B8. Controller B8 allows for either manual or computerized 
control. 
A sample well on plate B4, when containing upconverting phosphors, will 
emit visible light which is collected by a lens B9, passed through a 
filter B10, and focussed through a lens B11 and a shutter B12 onto PMT B3. 
PMT B3 outputs a current which is measured by a picoammeter B13. The PMT 
signal is proportional to the phosphor emission intensity. Shutter B12, 
controlled by a shutter driver B14, provides exposure protection to PMT 
B3, thereby preventing damage which may result from exposure to very 
intense light sources. Furthermore, overexposure of PMT B3 to light causes 
high dark currents which require several hours to decrease. PMT B3 is 
cooled for lower dark current and noise. Associated with the PMT cooler is 
a water-cooled power supply B15. A power supply B16 supplies high voltage 
to PMT B3. 
When the apparatus is operated in a computerized mode, a computer B17 
regulates controller B8 through an interface box B18. Picoammeter B13 can 
also be connected to computer B17, thereby allowing automated data 
acquisition to be performed. The data acquisition procedure moves 
translator B7 in the x direction to a first position at which location a 
specified number of current readings are taken and the average is 
calculated. Translator B7 then moves sample B4 a predetermined distance in 
the x direction to a new location where new data is collected. During this 
process, the data is plotted in order to provide the user with an 
immediate visual evaluation. After the scan is completed, the data can be 
saved or further data processing can be performed. FIG. 28 is an 
illustration of the data for upconverting phosphors in three test wells. 
FIG. 29 is a schematic view of a second embodiment of a hand-held probe for 
carrying out the present invention. This embodiment is comprised of a 
housing D1 and a capillary wick D2. Within housing D1 is a diode 
excitation laser D3, a lens assembly D4, a photodiode detector D5, and a 
battery supply D6. A display D7 mounted to one surface of housing D1 
communicates the results of the test to the user. In the preferred 
embodiment, laser D3 operates in the 960-980 nanometer range. 
In use, wick D2 wicks up a portion of a sample fluid D8 which is suspected 
of containing the target antigens. Target antigens bind to the antibodies 
present at a capture surface D9. Capture surface D9 is positioned at the 
focal point of source D3. The target antigens can be labeled with 
phosphor-antibody conjugates either before or after capture. In the 
preferred embodiment wick D2 is formed of glass. In this configuration 
capture surface D9 is prepared simply by filling the inside of the 
capillary with a bubble containing the antibodies of interest. By 
silanizing the inner surface with organofunctional silanes, conventional 
chemistries can be used to covalently link the antibodies or other 
biological macromolecule(s) to the inner tube wall at the site of the 
liquid bubble. The surface energy of the capillary is also easy to modify 
by silanization, which will help prevent nonspecific reagent and antigen 
adherence to the walls of the tube. 
In the preferred embodiment of this apparatus, the lower portion of wick D2 
is impregnated with upconverting phosphors that are conjugated to the 
target analytes or a crossreactive epitope for the capture probe. In use, 
the phosphor conjugates chromatograph towards capture surface D9 as sample 
fluid D8 is drawn up wick D2. As phosphors accumulate at capture surface 
D9, they will begin to emit visible light upon excitation by diode laser 
D3. The visible light emitted by the phosphors is detected by detector D5. 
The output of detector D5 is displayed on display D7. The amount of 
upconverted light reaching the detector is directly proportional to the 
concentration of labeled target antigen captured at the capture surface. 
The apparatus of FIG. 29 can be designed to simultaneously detect more than 
one target antigen. FIG. 30 illustrates a three channel configuration 
using interference filters. In this configuration capillary wick D2 is 
placed at the focus of a small parabolic reflector D10 capable of 
collecting approximately half of the emitted phosphorescence. The beam 
from diode laser D3 is directed onto capillary wick D2 at capture surface 
D9 along a direction perpendicular to the optical axis of reflector D10. 
Phosphorescent light from capture surface D9 is collected and collimated 
by mirror D10, directed through a notch filter D11 to reject the pump 
light, and onto three detectors D5 using three dichroic beam splitters 
D12. The reflectance bands of dichroic beamsplitters D12 are matched to 
the emission bands of the three phosphors used in the detection process. 
In an alternate embodiment of this apparatus, dichroic beamsplitters D12 
could be replaced with three bandpass filters used in the transmission 
mode. By placing the three filters on a rotation wheel, a single detector 
D5 could be used. Another alternative is to use a diffraction grating and 
a linear detector array to obtain an actual emission spectrum. 
FIG. 31A is an illustration of an embodiment of the invention in which-a 
diode laser array F1 and a detector array F2 are combined in a single 
device. In the preferred embodiment, arrays F1 and F2 are fabricated on a 
pair of silicon chips F3 with array dimensions of approximately 1 square 
centimeter. FIG. 31B is a detailed view of a small section of the device 
shown in FIG. 31A. Overlaying detector array F2 is a polymer film F4 of 
approximately 10 to 25 micrometers thickness which is used as the capture 
surface. Arrays F1 and F2 are separated by a spacer F5. Array F1 is 
comprised of Fabrey-Perot diode lasers, preferably tuned to 980 
nanometers. Lasers of this type are easily fabricated in gridded array 
patterns using conventional photolithography techniques. Each individual 
laser in array F1 has a columnar beam designed to strike only the adjacent 
portion of capture surface F4. The required power density of the 
individual lasers is dependent upon the efficiencies of the phosphors 
being used as well as the required detection efficiency. The detectors 
comprising array F2 are chosen to have an extremely low sensitivity in the 
wavelength region in which laser array F1 operates. If additional 
discrimination between the excitation and emission wavelengths is 
required, a cutoff filter can be used, preferably incorporated directly 
into capture surface F3. 
Upconverting phosphors F6 are conjugated by any of a variety of 
conventional biochemical crosslinking chemistries to antibody, nucleic 
acid probes, or other biological macromolecules (e.g., carbohydrates, 
lectins, streptavidin, MHC complexes), as well as to biological or 
chemical antigens (F7). Bonded to overlay F3 is a grid array F8 of 
complementary probes or antigens which are bound to capture surface F3 
using the same crosslinking chemistries. In use, a sample fluid F9 flows 
between arrays F1 and F2, target probes or antigens are captured by grid 
array F8 and excited by laser array F1, and the emissions detected by 
detector array F2. 
Typically, the upconverting phosphors to be used with this apparatus are 
approximately 0.1 to 0.5 micrometers. Since the size of the individual 
phosphor particles is of the order of the excitation wavelength, the power 
of the emission from the phosphors can be approximated by: 
EQU P.sub.em =Fnd.sup.3.4 I.sub.ex.sup.2 
where f is the phosphorescence efficiency (generally less than or equal to 
10.sup.-17 cm.sup.4 W.sup.-1 .mu.m.sup.-3.4 particle.sup.-1), N is the 
number of phosphor particles in the light path, D is the diameter of the 
phosphor particles, and I.sub.ex is the power density of the excitation 
source. 
Since the emitted power scales as the square of the excitation intensity, 
diagnostics using upconverting phosphors perform better in a microassay 
format. Assuming a constant power output from the excitation source, the 
excitation power density increases proportionally with the decrease in 
detection area, and the number of phosphor particles in the light path 
decreases linearly with a decrease in the detection area. Since the power 
of the light emitted from the phosphors scales with the square of the 
excitation power density, but linearly with the number of phosphors, 
P.sub.em will increase in inverse proportion to the detection area. 
Therefore, a 100.times.100 array will actually be 100 times more sensitive 
than a 10.times.10 array. 
Fluorescence-activated Cell Sorting 
The up-converting phosphors described herein can be used as phosphorescent 
labels in fluorescent cell sorting by flow cytometry. Unlike conventional 
fluorescent dyes, up-converting phosphors possess the distinct advantage 
of not requiring excitation illumination in wavelength ranges (e.g., UV) 
that damage genetic material and cells. Typically, upconverting phosphor 
labels are attached to a binding reagent, such as an antibody, that binds 
with high affinity and specificity to a cell surface protein present on a 
subset of cells in a population of cells in suspension. The 
phosphor-labeled binding component is contacted with the cell suspension 
under binding conditions, so that cells having the cell surface protein 
bind to the labeled binding reagent, whereas cells lacking the cell 
surface protein do not substantially bind to the labeled binding reagent. 
The suspended cells are passed across a sample detector under conditions 
wherein only about one individual cell is present in a sample detection 
zone at a time. A source, typically an IR laser, illuminates each cell and 
a detector, typically a photomultiplier or photodiode, detects emitted 
radiation. The detector controls gating of the cell in the detection zone 
into one of a plurality of sample collection regions on the basis of the 
signal(s) detected. A general description of FACS apparatus and methods in 
provided in U.S. Pat. Nos. 4,172,227; 4,347,935; 4,661,913; 4,667,830; 
5,093,234; 5,094,940; and 5,144,224, incorporated herein by reference. It 
is preferred that up-converting phosphors used as labels for FACS methods 
have excitation range(s) (and preferably also emission range(s)) which do 
not damage cells or genetic material; generally, radiation in the far red, 
and infrared ranges are preferred for excitation. It is believed that 
radiation in the range of 200 nm to 400 nm should be avoided, where 
possible, and the wavelength range 760 nm to 765 nm may be avoided in 
applications where maintenance of viable cells is desired. 
Additional Variations 
There are several apparatus design issues relating to the unique excitation 
and emission characteristics of upconverting phosphors which must be 
considered when using up-converting phosphors with flow cytometry. The 
first issue is the time required to reach maximum emission intensity. 
Since upconversion is a two photon process, upconverting phosphor emission 
is time delayed approximately 100 microseconds. The phosphor must remain 
within the excitation beam for this period of time regardless of the flow 
rate. Therefore given a flow rate between 1 and 10 meters per second with 
a channel width of 70 to 200 micrometers, the length of the excitation 
beam must be between 100 and 1000 micrometers. Given that the phosphor 
emissions saturate at an excitation intensity of about 200 watts per 
square centimeter, the laser source typically must have a power between 
0.01 and 400 milliwatts to achieve phosphor saturation. This implies that 
multiple laser diodes may be required to obtain maximum phosphorescence at 
the fastest flow rates. 
Another design issue is that associated with the detector. Since there is a 
considerable separation between the excitation and emission wavelengths of 
the upconverting phosphors, detection can be performed using a 
photomultipler tube (PMT), a photodiode, or a CCD array. The 
phosphorescence decay time is long, with a decay half life of 
approximately 300 microseconds. The most sensitive method of detection is 
to integrate the signal measured by the PMT. However, 99 percent detection 
of the available phosphorescent signal requires that the phosphor remain 
in the sight path of the detector for 5 times the phosphorescence decay 
half-life (i.e., 1.5 milliseconds). Assuming a flow rate of 10 meters per 
second and a channel width of 200 micrometers, the PMT must be able to 
detect over a path length of 1.5 centimeters. This path length is also the 
required spacing between cells flowing through the cytometer, implying a 
maximum count rate of 667 cells per second. It is, however, possible to 
sacrifice some detection sensitivity by reducing the detection path 
length, at least to that required to attain steady-state emission from the 
phosphors. As long as a steady-state emission peak is reached by the 
phosphor in the excitation window, the peak signal received by the PMT 
should be directly proportional to the concentration of phosphors present. 
The nonphotobleaching property of the phosphors makes this form of 
detection possible. The loss in detection sensitivity corresponding to a 
0.1 centimeter path length (versus a 1.5 centimeter path length) is 
approximately a factor of 3. Triggering the emission detector can be 
accomplished by observing the light scattered by the cell as it passes 
through the excitation source. 
In environments where absorption of the up-converted phosphor radiation is 
high, the phosphor microparticles are coated with a fluorescent dye or 
combination of dyes, in selected proportions, which absorb at the 
up-converted frequency and subsequently re-radiate at other wavelengths. 
Because the single-photon absorption cross-sections for these fluors are 
typically very high, only a thin layer is required for complete absorption 
of the phosphor emission. This coat particle may then be encapsulated and 
coated in a suitable antigen or antibody receptor (e.g. microparticle). An 
example of this layering is depicted schematically in FIG. 9. There exists 
a wide variety of fluorescent dyes with strong absorption transitions in 
the visible, and their emission covers the visible range and extends into 
the infrared. Most have fluorescent efficiencies of 10% or more. In this 
manner, the emission wavelengths may be custom-tailored to pass through 
the particle's environment, and optical interference filters may again 
used to distinguish between excitation and emission wavelengths. If a 
relatively large wavelength "window" in the test medium exists, then the 
variety of emission wavelengths which may be coated on a single type of 
phosphor is limited only by the number of available dyes and dye 
combinations. Discrimination between various reporters is then readily 
carried out using the spectroscopic and multiplexing techniques described 
herein. Thus, the number of probe/reporter "fingerprints" which may be 
devised and used in a heterogenous mixture of multiple targets is 
virtually unlimited. 
The principles described above may also be adapted to driving 
species-specific photocatalytic and photochemical reactions. In addition 
to spectroscopic selection, the long emission decay times of the phosphors 
permit relatively slow reactions or series of reactions to take place 
within the emission following photoexposure. This is especially useful 
when the phosphor-catalyst or reactant! conjugate enters an environment 
through which the excitation wavelength cannot penetrate. This slow 
release also increases the probability that more targets will interact 
with the particle. 
The unique decay rates of phosphor particles allow dynamic studies as well. 
In a system where continuous exposure to the excitation source is not 
possible, or is invasive and thereby undesirable, pulsed excitation 
followed by delayed fluorescence detection is necessary. After the 
phosphor reporter has been photoexcited, the subsequent emission from the 
phosphor or phosphor/dye conjugate particle lasts typically about a 
millisecond. In a dynamic environment, such as a static or flowing system 
with moving targets, the particle will emit a characteristically decaying 
intensity of light as it travels relative to the excitation/detection 
apparatus. Combined with imaging optics appropriate to the scale of the 
system and the velocities within the system, a CCD photoelectric sensor 
array will be used to detect the particle or particles movement across the 
array's field of view. The delayed emission of the phosphors, which is a 
well-characterized function of time, makes possible the dynamic tracking 
of individual particle's positions, directions and velocities, and 
optionally calculation of particle size, density, and hydrodynamic 
conformation. As a particle moves, it exposes more elements of the array, 
but with every-decreasing intensity. The more elements it exposes over a 
certain fraction of its decay time, the faster it is moving. Therefore, 
the integrated intensity pattern of a particle's emission "track" 
collected by the array is directly related to the velocity of the 
particle. The particles may be refreshed again at any time by the pulsed 
or chopped CW excitation source. FIG. 10 illustrates this scheme. Although 
only a depiction of "side-on" excitation and detection is shown, both 
side-on and end-on detection and excitation arrangements, or combinations, 
are possible. Reduction of the CCD array intensity information by computer 
analysis will allow near-real time tracking of the particles in a 
dynamically evolving or living systems. Data analysis and reduction 
performed by the computer would include a convolution of the intrinsic 
decay of the phosphor emission, the number of pixels illuminated and their 
signal level, the orientation of the decaying signal on the array, and the 
intensity contributions from a blur circle from particles moving in and 
out of the focal plane of the array. In an end-on flow detection 
arrangement, the size of the blur circle would relate directly to how 
quickly the particle moves out of focus, thereby allowing the velocity of 
the particle to be determined. One possible application would be 
monitoring the chemistry and kinetics in a reaction column, alternatively, 
the application of this method to flow cytometry may permit the resolution 
of cells on the basis of hydrodynamic properties (size, shape, density). 
The method may also be useful for in vivo diagnostic applications (e.g., 
blood perfusion rate). 
Up-converting phosphor labels may also be used to sense the temperature in 
the region at which the up-converting phosphor label is bound. 
Up-converting phosphor temperature measurement methods are described in 
Berthou H and Jorgensen CK (October, 1990) Optics Lett. 15(19): 1100, 
incorporated herein by reference. 
Although the present invention has been described in some detail by way of 
illustration for purposes of clarity of understanding, it will be apparent 
that certain changes and modifications may be practiced within the scope 
of the claims. 
The broad scope of this invention is best understood with reference to the 
following examples, which are not intended to limit the invention in any 
manner. 
EXPERIMENTAL EXAMPLES 
Validation of Up-Converting Inorganic Phosphors as Reporters 
Up-converting phosphor particles comprising sodium yttrium fluoride doped 
with ytterbium-erbium were milled to submicron size, fractionated by 
particle size, and coated with polycarboxylic acid. Na(Y.sub.0.80 
Yb.sub.0.18 Er.sub.0.02)F.sub.4 was chosen for its high efficiency upon 
excitation in the range 940 to 960 nm. A Nd:Yag pumped dye laser/IR dye 
combination was used to generate 8-ns to 10-ns duration pulses in the 
above frequency range. 
The laser pulses were used to illuminate a suspension of milled phosphor 
particles in liquid and attached to glass slides in situ. The suspension 
luminescence observed at right angles was monitored using a collection 
lens, a spatial filter in order to filter out scattered excitation light 
to the maximum possible extent, and a photomultiplier, vacuum photodiode, 
or simple solid state photodiode (depending on the light level observed). 
The luminescent signal level was determined as a function of solution pH 
(range: 6-8), grain size, particle loading (.lambda.g/cm.sup.3), and the 
nature of stabilizing anionic surfactant. Signals were recorded both as a 
time integral from a boxcar integrator and from a long RC time constant or 
as a transient signal using a transient digitizer in order to delineate 
the luminescence lifetime under particular experimental conditions. In 
situ signals were also measured by laser scanning microscopy. FIG. 11 is a 
fluorescence scan of the phosphor emission spectrum incident to excitation 
with a laser source at a wavelength maximum of 977.2 nm; emission maximum 
is about 541.0 nm. FIG. 12 is an excitation scan of the phosphor 
excitation spectrum, with emission collection window set at 541.0 nm; 
excitation maximum for the phosphor at the 541.0 nm, emission wavelength 
is approximately about 977 nm. FIG. 13 is a time-decay measurement of the 
phosphor luminescence at 541.0 nm after termination of excitation 
illumination; maximal phosphorescence appears at approximately 400 .mu.s 
with a gradual decay to a lower, stable level of phosphorescence at about 
1000 .mu.s. FIG. 14 shows the phosphor emission intensity as a function of 
excitation illumination intensity; phosphorescence intensity increases 
with excitation intensity up to almost about 1000 W/cm.sup.3. 
Phosphorescence efficiencies of submicron Na(Y.sub.0.8 Yb.sub.0.12 
Er.sub.0.08)F.sub.4 particles were measured. A Ti:sapphire laser was used 
as an excitation source and a spectrophotometer and photomultiplier was 
used as a detection system. Two types of measurement were performed. The 
first was a direct measurement in which the absolute emission per particle 
for phosphor suspensions was measured in emission bands at 540 nm and 660 
nm. The calibrated cross-sections are shown in FIG. 15, and 
size-dependence is shown graphically in FIG. 16. This corresponded to a 
phosphorescence cross-section of approximately 1.times.10.sup.-16 cm.sup.2 
for 0.3 .mu.m particles with excitation light at 975 nm and an intensity 
of approximately 20 W/cm.sup.2. The emission efficiency of dry phosphor 
powder of about 25 .mu.m was also measured. On the basis of known values 
for the absorption cross-section of Yb.sup.+3 in crystalline hosts 
(Lacovara et al. (1991) Op. Lett. 16: 1089, incorporated herein by 
reference) and the measured dependence of the phosphorescence emission on 
particle size, a phosphorescence cross-section of approximately 
1.times.10.sup.-15 cm.sup.2 was found. The difference between these two 
measurements may be due to a difference in phosphorescence efficiency 
between dry phosphor and aqueous suspensions, or due to absorption of 
multiply scattered photons in the dry phosphor. On the basis of either of 
these cross-section estimates, the cross-section is sufficiently large to 
allow detection of single submicron phosphor particles at moderate laser 
intensities. At laser intensities of roughly 10 W/cm.sup.2, the 
phosphorescence scales as the laser intensity to the 1.5 power. 
Phosphor Particle Performance: Sensitivity of Detection 
A series of Terasaki plates containing serial dilutions of monodisperse 0.3 
.mu.m up-converting phosphor particles consisting of (Y.sub.0.86 
Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.2 S were tested for up-conversion 
fluorescence under IR diode laser illumination in a prototype instrument. 
The phosphor particles were prepared by settling in DMSO and were serially 
diluted into a 0.1% aqueous gum arabic solution. This appeared to 
completely eliminate any water dispersion problems. The serial dilutions 
used are listed in Table III. 
TABLE III 
______________________________________ 
Equivalent 
Phosphor Phosphor Detection 
Loading Loading Sensitivity 
Label (ng/well) (particles/well) 
(M) 
______________________________________ 
10.sup.0 
1700 .+-. 90 23,600,000 .+-. 1,200,000 
4 .times. 10.sup.-12 
10.sup.-1 
170 .+-. 9 2,360,000 .+-. 120,000 
4 .times. 10.sup.-13 
10.sup.-2 
17 .+-. 0.9 236,000 .+-. 12,000 
4 .times. 10.sup.-14 
10.sup.-3 
1.7 .+-. 0.09 
23,600 .+-. 1,200 
4,10.sup.-15 
10.sup.-4 
0.170 .+-. 0.009 
2,360 .+-. 120 4 .times. 10.sup.-16 
10.sup.-5 
0.017 .+-. 0.0009 
236 .+-. 12 4 .times. 10.sup.-17 
10.sup.-6 
0.0017 .+-. 0.00009 
23.6 .+-. 1.2 4 .times. 10.sup.-18 
______________________________________ 
The stock DMSO dispersion had a phosphor density of 1.70.+-.0.09 mg/mL (at 
95% confidence limits), determined gravimetrically by evaporating 4-1 mL 
samples. This translates to 23.6.times.10.sup.9 particles/mL (assuming an 
average particle size of 0.3 .mu.m and particle density of 5.3 g/mL). The 
residue after evaporating the samples over the weekend at 
110.degree.-120.degree. C . was noticeably yellow, but did phosphoresce 
when tested with an IR diode laser. 
Visual green light emanated from all serial dilutions down to 10.sup.-3 
(i.e., 1.7 .mu.g/mL or 23.6.times.10.sup.6 particles/mL) in a 1 mL 
polypropylene microfuge tube using a hand-held diode laser in a dark room. 
The 10.sup.-1 and 10.sup.-2 dilutions were visibly cloudy. Either 1 .mu.l 
of each serial dilution, or 0.1 .mu.l of the next higher dilution, were 
pipetted into a well on the Terisaki plate. It was found that 1 .mu.l 
fills the bottom of the well and 0.1 .mu.l spreads along the edge of the 
well, but does not cover the entire surface. Because of the statistical 
and pipetting problems associated with small volumes with low particle 
concentrations, 2 to 4 replicates were prepared of each dilution. 
The well of a Terasaki plate holds a 10 .mu.l sample volume. Assuming all 
the phosphor particles contained in this volume adhere to the bottom of 
the sample well, we can estimate an equivalent detection sensitivity 
(Table III). It should be noted that 10.sup.-15 to 10.sup.-18 M is the 
normal range of enzyme-linked surface assays. 
Control Sample Results 
The control samples were scanned using a prototype up-conversion 
fluorimeter device (David Sarnoff Research Center). The samples were 
scanned by moving the plate in 50 .mu.m increments, using a motorized X-Y 
positioning stage, relative to the focal point of an infrared diode laser. 
The IR diode laser was operated at 63 mW (100 mA). The beam was focused to 
2.4.times.10.sup.-3 cm.sup.2 at the focal point. As the bottom of the 
sample well is about 1.4.times.10.sup.-2 cm.sup.2 (1365 .mu.m diameter), 
the beam covers less than 17% of the well bottom surface at any individual 
position. The well also has sloping side walls which widen from bottom to 
top of the sample well and are also interrogated by a progressively 
divergent laser beam. Neglecting losses in the optics, the IR light 
intensity at the focal point (bottom of the sample well) was approximately 
26-27 W/cm.sup.2 at 980 nm wavelength. A photomultipler tube (PMT) was 
used for detection of the visible (upconverted) light emitted from the 
sample. Since the laser beam width was smaller than the surface area at 
the bottom of the sample well, the plate was aligned by visual inspection 
against the focal point of the diode laser so that the laser was centered 
in the middle well (C6 when reading wells C5, C6 and C7, and D6 when 
reading wells D5, D6, and n7). 
The PMT signal (amps) was recorded at each plate position and numerically 
integrated over the width of the sample well (approximately 4000 .mu.m). 
Several scans were made at different positions in the 10.sup.-2 to 
10.degree. dilution sample wells to determine the uniformity of the 
particle distribution. The background signal was determined by integrating 
the average dark field current of the PMT over a 4000 .mu.m distance, 
which yields an integrated background signal of 1.times.10.sup.-9 .mu.a-m. 
The integration products of the samples wells were scaled to this 
background signal, and are shown in FIG. 19. 
Immunodiagnostic Sample Detection 
A series of IgG/anti-IgG samples for demonstrating the capabilities of the 
up-converting phosphor reporters in a immunosorbant assay format was 
prepared. These samples consisted of six individual wells (positive 
samples) coated with antigen (mouse IgG) and bovine serum albumin (BSA), 
and six wells coated with BSA alone (negative controls). Nominal 0.3 .mu.m 
(Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.2 S phosphor particles 
coated with goat anti-mouse IgG antibody (anti-IgG) were then used as the 
reporter-antibody conjugate. 
Six wells (C5, C6, C7, D5, D6, and D7) of a clear polystyrene Terasaki 
plate were coated with mouse IgG by incubating at 37.degree. C. against 5 
.mu.L of a 100 .mu.g/.mu.L mouse IgG solution in phosphate buffered saline 
(PBS). After 1 h, this solution was aspirated off and each sample well was 
washed with 10 .mu.L of 3% BSA in PBS. This was immediately aspirated off 
and replaced with 20 .mu.L of 3% BSA in PBS. Each sample well was 
post-coated with BSA by incubating against the 20 .mu.L of BSA/PBS 
solution for 1 h at 37.degree. C. The post-coat solution was aspirated off 
and the plates stored at 4.degree. C. overnight. These wells were 
considered in positive samples. The same six wells in a second Terasaki 
plate were prepared in an identical fashion, except they were not coated 
with mouse IgG. This second set of sample wells were considered negative 
controls. 
Phosphor-Antibody Conjugate 
A solution of (Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.2 S phosphor 
particles was prepared by suspending the dry phosphors into DMSO. The 
initial particle density was approximately 10.sup.7 particles/mL as 
determined by counting the number of particles contained in the field of 
an optical microscope. It should be noted that the 0.3 .mu.m fundamental 
particle size was below the resolution limits of the microscope. This 
solution was allowed to settle undisturbed for 3 days. The supernatant, 
which was turbid and presumably contained mostly monodisperse smaller 
particles was used for subsequent conjugation. 
Goat anti-mouse IgG antibody (Ab) was conjugated (by adsorption) onto the 
DMSO fractionated phosphor particles. This was done by mixing 200 .mu.L of 
the Ab solution (in 0.1M Tris-HCl, pH 7.2) with 100 .mu.L of the phosphor 
suspension in DMSO. Several different Ab concentrations were tried in the 
range of 0.025 to 1 .mu.g/.mu.L. A concentration of 0.25 .mu.g/.mu.L 
appeared to result in the most efficient coating (i.e., maximum Ab 
utilization with a minimum of clumping of the phosphor particles). The 
phosphors were equilibrated overnight at room temperature with the Ab in 
this DMSO/Tris solution with gentle agitation. The resulting phosphor-Ab 
conjugates were centrifuged from this solution and resuspended in a 3 
.mu.g/mL BSA solution in PBS for post-coating. The resulting BSA/PSA 
resuspension was used directly for the assay. 
The degree of Ab adsorption to the phosphors, and residual Ab activity, was 
determined by titrating the phosphor-bound Ab with a fluorescein 
isothiocyanate (FITC) conjugated-mouse IgG. The resulting FITC-labeled 
phosphors were passed through a Cyteron Absolute flow cytometer, which was 
also capable of measuring the relative size of the particles. Two distinct 
size subpopulations were observed with about 65% of the counted particles 
appearing as small, presumably monodisperse particles, and 35% being 
significantly larger, presumably aggregates. Only 60% of the smaller 
subpopulation appeared to have significant quantities of active Ab 
(determined by FITC fluorescence). Of the purported aggregates, about 90% 
appeared to contain active Ab (by FITC fluorescence). This suggests that 
less than 40% of the phosphor-Ab conjugates were of an appropriate size 
(nominal 0.3 .mu.m) and exhibited anti-mouse IgG activity. A similar 
fraction of phosphor-Ab conjugates (31%) were active but carried a 
significantly larger phosphor reporter. 
The PMT signal (amps) was recorded at each plate position and numerically 
integrated over the width of the sample well (approximately 4000 .mu.m). 
The average signals (with 95% confidence limits) are: 
Average of Positive Samples=1.30.times.10.sup.-4 .+-.1.25.times.10.sup.-4 
.mu.a-m 
Average of Negative Controls=4.20.times.10.sup.-6 .+-.6.82.times.10.sup.-6 
-a-m 
The positive samples and negative controls are statistically different at 
the 99.9% confidence level. The positive samples emit on average 
30.0.+-.29.7 times more light than the negative controls. 
Linkage of Phosphors to Biological Macromolecules 
In order to delineate further the parameters for up-converting phosphors as 
biochemical reporters, biological linkers were attached to phosphor 
particles. Sodium yttrium fluoride-ytterbium/erbium phosphor particles 
were coated with streptavidin. The excitation and emission spectral 
properties of the phosphor alone and the phosphor coated with streptavidin 
were measured (FIGS. 17A, 17B, 18A, and 18B) and both the uncoated and 
streptavidin-coated phosphors were almost identical in their absorption 
and emission properties, indicating that the attachment of macromolecular 
linkers (e.g., proteins) have little if any effect on the phosphorescent 
properties of the up-converting phosphor. The streptavidin-coated 
phosphors were then specifically bound to biotinylated magnetic beads, 
demonstrating the applicability of linker-conjugated inorganic phosphors 
as reporters in biochemical assays, such as immunoassays, 
immunohistochemistry, nucleic acid hybridizations, and other assays. 
Magnetic bead technology allows for the easy separation of biotin-bound 
streptavidin-coated phosphor from a solution, and is particularly 
well-suited for sandwich assays wherein the magnetic bead is the solid 
substrate. 
Advantageously, streptavidin-biotin chemistry is widely used in a variety 
of biological assays, for which up-converting phosphor reporters are 
suited. FIG. 20 shows schematically, for example and not limitation, one 
embodiment of an immunoassay for detecting an analyte in a solution by 
binding the analyte (e.g., an antigen target) to a biotinylated antibody, 
wherein the analyte forms a sandwich complex immobilized on a solid 
substrate (e.g., a magnetic bead) by linking a first binding component 
bound directly to the solid substrate to a second binding component (e.g., 
the biotinylated antibody); a streptavidin-coated up-converting phosphor 
then binds specifically to the biotinylated antibody in the sandwich and 
serves to report formation of the sandwich complex on the solid substrate 
(which is a measure of the analyte concentration). When the solid 
substrate is a magnetic bead, it is readily removed from the sample 
solution by magnetic separation and the amount of phosphor attached to the 
bead(s) in sandwich complex(es) are determined by measuring specific 
up-converting phosphorescence. Thus, sandwich complex phosphorescence 
provides a quantitative measure of analyte concentration. 
Biotinylated polynucleotides are also conveniently used as hybridization 
probes, which can be bound by streptavidin-coated up-converting phosphors 
to report hybrid formation. 
Background Phosphorescence in Biological Samples 
Background signals were determined in two biological samples for 
determination of potential background in immunoassays. Sputum and urine 
were used as samples in the same apparatus as used for the phosphorescence 
sensitivity measurements (supra). No background levels were found above 
the system noise levels set by the photomultiplier dark current. This 
noise level allows detection of signals from on the order of a few hundred 
particles/cm.sup.3. This is close to a single particle in the detection 
volume of the system. 
A photomultiplier is a preferred choice for a detector for high sensitivity 
measurements of up-converting phosphors since photomultipliers can be 
selected to produce high quantum efficiency at the up-converted (i.e., 
emitted) wavelengths and virtually no response in the range of the longer 
excitation wavelengths. 
Detection of Cell Antigens with Phosphor-Labeled Antibodies 
Streptavidin is attached to the up-converting phosphor particles as 
described, supra. The mouse lymphoma cell line, EL-4, is probed with a 
hamster anti-CD3 antibody which specifically binds to the 30 kD cell 
surface EL-4 CD3 T lymphocyte differentiation antigen. The primary hamster 
antibody is then specifically bound by a biotinylated goat-antihamster 
secondary antibody. The biotinylated secondary antibody is then detected 
with the streptavidin-phosphor conjugate. This type of multiple antibody 
attachment and labeling is termed antibody layering. 
Addition of multiple layers (e.g., binding the primary hamster Ab with a 
goat-antihamster Ab, followed by binding with a biotinylated 
rabbit-antigoat Ab) are used to increase the distance separating the 
phosphor from the target. The layering effect on signal intensity and 
target detection specificity is calibrated and optimized for the 
individual application by performing layer antibody layering from one 
layer (primary antibody is biotinylated) to at least five layers and 
ascertaining the optimal number of layers for detecting CD3 on EL-4 cells. 
FIG. 21 schematically portrays simultaneous detection of two EL-4 cell 
surface antigens using phosphors which can be distinguished on the basis 
of excitation and/or emission spectra. Detection of both antigens in the 
scheme shown in FIG. 21 uses a biotinylated terminal antibody which is 
conjugated to streptavidin-coated phosphor (#1 or #2) prior to incubation 
with the Ab-layered sample. Thus, the phosphor-antibody specificity is 
retained through the unusually strong (K.sub.D approx. 1.times.10.sup.15 
M.sup.-1) non-covalent bond between streptavidin and biotin which is 
pre-formed before incubation with the primary antibody-bound sample. 
Quantitation of each antigen is accomplished by detecting the distinct 
signal(s) attributable to each individual phosphor species. Phosphorescent 
signals can be distinguished on the basis of excitation spectrum, emission 
spectrum, fluorescence decay time, or a combination of these or other 
properties. 
FIG. 22 shows a schematic of an apparatus for phase-sensitive detection, 
which affords additional background discrimination. The pulse or frequency 
mixer is set to pass the signal and discriminate against the background 
following frequency calibration for maximum background rejection. 
Covalent Conjugation of Upconverting Phosphor Label to Avidin 
An upconverting ytrium-ytterbium-erbium (Y.sub.0.86 Yb.sub.0.08 
Er.sub.0.06) oxysulfide (O.sub.2 S) phosphor was linked to avidin by the 
following procedure: 
Monodisperse upconverting phosphor particles were silanized with 
thiopropyltriethoxysilane (Huls) following the procedure detailed by 
Arkles (in: Silicone Compounds: Register and Review, Hills America, pgs. 
59-75, 1991). This consisted of adding thiopropyltriethoxysilane (2 g) and 
95% aq. ethanol (100 mL) to a 500 mL Erlenmeyer flask and stirred for 2 
minutes. Approximately 8 mL of the 65 mg/mL phosphor suspension in DMSO 
was then added to the mixture. This suspension was stirred for an 
additional 2 minutes, then transferred to centrifuge tubes and centrifuged 
to separate the phosphor particles. The pellets were washed twice with 95% 
aq. ethanol centrifuging each time. The resulting particles were collected 
and dried overnight under vacuum at approximately 30.degree. C. A quantity 
(127 mg) of dry silanized phosphors were resuspended in 1.5 mL of DMSO 
(phosphor stock). 
A solution containing 1.19 mg of avidin (Pierce) in 1.0 mL of borate buffer 
(954 mg sodium borate decahydrate and 17.7 mL of 0.1N NCl in 50 mL of 
deionized water, pH 8.3) was prepared (Avidin stock). Another solution 
containing 1.7 mg of N-succinimidyl(4-iodoacetyl) aminobenzoate (Pierce 
Chemical) in 1.2 mL of DMSO was prepared (SIAB stock). A quantity (10 
.mu.L) of the SIAB stock was added to the 1.0 mL of Avidin stock and 
stirred at room temperature 30 min to allow the N-hydroxysuccimide ester 
of the SIAB to react with primary amines on the avidin (Avidin-SIAB 
stock). 
A 20 mL scintillation vial was prepared containing 10 mL of borate buffer 
(pH 8.3). The following additions were then made to this vial: 21.6 .mu.L 
of the avidin-SIAB stock solution followed by 1.5 mL of the phosphor 
stock. This reaction mixture was stirred at room temperature in the dark 
overnight to allow the SIAB activated avidin to react with the thiol 
groups present on the silanized phosphor surface and resulting in the 
covalent linkage of avidin to the phosphor particles. 
After the overnight incubation 1.0 mL of the reaction mixture was 
centrifuged (1 min at 10,000 g) and the supernatant removed. The pellet 
was resuspended in 1.0 mL of phosphate buffered saline (pH 7.2, Pierce) 
and centrifuged again to wash any unconjugated protein from the phosphors. 
This washing process was repeated. The washed pellet was resuspended in 
1.0 mL of phosphate buffered saline and used directly in diagnostic assays 
as described below. 
Measurement Apparatus 
A modified SLM Aminco 48000 Fluorimeter was used to measure the 
fluorescence spectrum from the phosphor samples. The modifications to this 
device consisted of adding a laser diode (David Sarnoff CD-299R-FA #13) 
which was input to the fluorimeter through port 3. The laser diode emits 
at .lambda.=985.1 nm. Spectral data provided by the David Sarnoff Research 
Center also shows a small peak at 980.2 nm. This peak has 15% the 
intensity of the peak at 985 nm. 
A 5.08 cm focal length lens was used to collimate the diode laser beam. The 
power of the IR laser light was measured as 6.1 mW at the cuvette location 
with a drive current of 75 mA. The beam was not focused at the center of 
the cuvette. This is true for the standard visible light from the 
fluorimeter excitation monochromator as well. The laser diode beam is 
diverging as it enters the cuvette holder and is approximately 4 mm 
(H).times.2 mm (V) by the time it reaches the center of the cell, 
neglecting the changes in refractive index of the cell wall and the 
liquid. 
Light emitted is scanned with a monochromator and detected by a 
photomultiplying tube (PMT) 90.degree. from the direction of the 
excitation light. The detection limits for the modified SLM Aminco 48000 
were determined by serial dilution to be 4.times.10.sup.-16 M (240,000 
phosphor particles per mL) in PBS. Phosphor emission peaks in the spectrum 
were seen at wavelengths of 406.+-.2 nm, 434.+-.2 nm, 522.+-.2 nm, and 
548.+-.2 nm. The largest peak was at 548 nm. The intensity of the 548 nm 
peak was used to discriminate samples. 
Linkage of Avidin-Phosphor Conjugate to Cell Surface Marker 
A lymphoblastoid cell line (Human Genetic Mutant Cell Repository #GM07092) 
was cultured in RPMI 1640 media containing 15% heat inactivated fetal calf 
serum. A suspension of cells (10.sup.7 cells) was centrifuged and 
resuspended in an equal volume of phosphate buffered saline (PBS) pH 7.4. 
Cells were washed two times in PBS and resuspended to a final 
concentration of 5.times.10.sup.6 cells/mi. These cells were then 
incubated with a mouse IgGl monoclonal antibody to human .beta..sub.2 
-microglobulin, a Class I histocompatibility antigen in polystyrene 
centrifuge tubes. The cells were immunoprecipitated for 30 minutes at 
4.degree. C. with an antibody concentration of 10 .mu.g/ml. The cells were 
harvested by centrifugation, washed twice in PBS, resuspended in PBS and 
then aliquoted (250 .mu.L) into six fresh centrifuge tubes. Four of these 
samples received biotinylated goat antimouse IgG, while the remaining two 
received FITC-labelled goat anti-mouse IgG. These immunoprecipitations 
were performed at 4.degree. C. for 30 minutes in volume of 400 .mu.L with 
a final second antibody concentration of 20 .mu.g/ml. The cells were 
harvested and washed in PBS as above but were resuspended in 50 .mu.L of 
blocking buffer (0.2% purified casein in PBS, Tropix, Bedford, Mass.). The 
cell-antibody complexes were blocked in this solution for 30 minutes at 
room temperature and then transferred to fresh tubes. 
A pre-blocked suspension (40 .mu.L) of either avidin-Phosphor conjugate, 
avidin-FITC, avidin, or unconjugated Phosphor was added to four of the 
cell samples conjugated with the biotinylated anti-mouse IgG (H&L). In 
addition, an equal amount of pre-blocked avidin-Phosphor or unconjugated 
Phosphor was added to the remaining two cell samples immunoprecipitated 
with the non-biotinylated FITC-labelled anti-mouse IgG (H&L). The avidin 
reporter conjugates or negative controls were preblocked as follows. 
Avidin-Phosphor and Phosphor alone was diluted in blocking buffer by 
adding 10 .mu.L of a 6.7 mg/ml suspension to a final volume of 100 .mu.L. 
Avidin-FITC and the avidin alone controls were also diluted in blocking 
buffer by adding 27 .mu.L of 2.5 mg/ml solution to a final volume of 100 
.mu.L. These reagents were blocked at room temperature for 3 hours with 
intermittent resuspension and then added to 50 .mu.L of cells labelled 
with biotinylated or non-biotinylated second antibody. The avidin-biotin 
reactions were performed at room temperature for 30 minutes with 
occasional resuspension. The reactions were stopped by harvesting the 
cells by centrifugation and washing twice in blocking buffer. The samples 
were resuspended in 100 .mu.L of blocking buffer and allowed to settle for 
4-5 minutes. Slides for imaging were prepared by pipetting 5 .mu.L of 
settled cells from the bottom of the tube. Cells were imaged by confocal 
laser microscopy under appropriate conditions to observe cell surface FITC 
and upconverting phosphor signals. The observations are summarized in 
Table IV. 
TABLE IV 
______________________________________ 
Cell Cell 
Surface 
Surface 
Type of goat 
Avidin Phosphor 
FITC 
Tube anti-mouse IgG 
Conjugate Signal Signal 
______________________________________ 
1 biotinylated 
Avidin-Phosphor 
+ - 
2 biotinylated 
Phosphor - - 
3 biotinylated 
Avidin - - 
4 biotinylated 
Avidin-FITC - + 
5 FITC labelled 
Avidin-Phosphor 
- + 
6 FITC labelled 
Phosphor - + 
______________________________________ 
The remainder of the samples were used to resuspend paramagnetic, 
polystyrene beads bound with sheep anti-mouse IgG. For each of the six 
samples, 3.times.10.sup.7 beads were pre-washed with blocking buffer for 1 
hour at room temperature in Eppendorf tubes. The buffer was removed by 
aspiration while the tubes were in a magnetic rack. The magnetic beads 
with anti-mouse IgG were allowed to bind to the antibody labelled cells 
for 1 hour at room temperature with intermittent resuspension. The 
magnetic beads were then collected on a magnetic rack, washed four times 
in blocking buffer, resuspended in 100 .mu.L blocking buffer, transferred 
to a fresh tube, and up-converting phosphorescence was measured on the 
fluorimeter. 
To scan for phosphor emission, the emission monochromator bandwidth was set 
to 8 nm and the spectra were scanned from 500 to 700 nm with a step size 
of 2 nm. Samples were also measured for FITC signal by exciting the 
samples with 37 .mu.M at .lambda.=490 nm with a 2 nm bandwidth. Since the 
excitation wavelength (490 nm) and the emission wavelength (514 nm) are 
very close for FITC, higher resolution was required to get separable 
signals than with phosphor labelling. The intensity of the 490 nm signal 
was 240 .mu.W/cm.sup.2 at the center of the well. FITC emission spectra 
were scanned at 0.5 nm increments from 450 nm to 750 nm with a 2 nm 
bandwidth on the emission monochromator. Sample 1 is the positive control 
and clearly yielded the highest emission signal. Sample 2 indicates that 
any nonspecific adsorption of the phosphors to the sample is limited and 
is readily discriminated from signal attributable to avidin-conjugated 
phosphor and showing that avidin linked phosphors can specifically bind 
only when they are conjugated with the probe, in this example through the 
biotin-avidin linkage. Sample 3 is the negative control which contains no 
phosphors, only avidin. Sample 4 shows FITC-conjugated avidin. Although 
FITC signals were observed on the cell surface by laser microscopy, the 
signals were below the level of detection on the fluorimeter for 
measurement of FITC, and since there was no phosphor in the sample there 
was no significant phosphor signal. Samples 5 and 6 show that 
FITC-conjugated primary antibodies can be detected and that the presence 
of phosphor or avidin-phosphor does not significantly disrupt binding of 
the primary antibody to its target antigen. 
Linkage of Avidin-Phosphor Conjugate to DNA 
Plasmid DNA (25 .mu.g) was nick translated in the presence of 20 mM dGTP, 
20 mM dCTP, 20 mM biotin-14 dATP, 13 mM dTTP, and 7 mM digoxigenin-11 dUTP 
and purified by ethanol precipitation. The average size of the 
biotinylated, digoxygenin labelled fragments was estimated to be between 
200-300 nucleotides as estimated by gel electrophoresis. Approximately 20 
.mu.g DNA was immunoprecipitated for 1 hour at 22.degree. C. with 10 
.mu.g/ml mouse monoclonal anti-digoxigenin IgGl solution (PBS) in a 200 
.mu.L volume. An equivalent reaction containing no DNA was also prepared. 
Each of the two samples were then aliquoted (50 .mu.L) into three fresh 
Eppendorf tubes. 
The avidin-conjugates were blocked for 1 hour at room temperature by 
diluting 500 .mu.g of an avidin-phosphor suspension, unconjugated phosphor 
suspension, or avidin solution in 300 .mu.L of blocking buffer. For each 
of the samples (summarized below in Table V) 50 .mu.L of the 
anti-digoxigenin conjugates was added to 150 .mu.L of pre-blocked 
avidin-conjugates or avidin and were incubated for 30 minutes at room 
temperature. 
Unbound avidin-conjugates were removed by resuspending 3.times.10.sup.7 
paramagnetic beads linked with sheep anti-mouse IgG (pre-blocked in 
blocking buffer). After incubation for 30 minutes at room temperature with 
intermittent resuspension, the beads were separated on a magnetic rack and 
washed 4 to 6 times in PBS. The antibody-DNA bound beads were then 
measured on the fluorimeter. 
The samples were scanned from 500 to 700 nm with a bandwidth of 8 nm and 
step size of 2 nm. Each PMT value reported (Table V) represents an average 
over 5 scans. Sample 1 is expected to provide the highest PMT signal since 
biotinylated DNA is present and can bind to the avidin-linked phosphors. 
Sample 2 indicates the level of nonspecific adsorption of the phosphors to 
the sample which is found to be insignificant since the PMT signal is 
observed to be the same as that of the negative control (sample 4) which 
contains no phosphors. Sample 3 is another control and shows that the 
avidin-linked phosphors do not bind to the paramagnetic beads in the 
absence of DNA. Samples 5 and 6 show results of FITC-labeled avidin used 
to validate assay. 
TABLE V 
______________________________________ 
Upconverting Phosphor Nucleic Acid Diagnostic Assay Results 
PMT Signal 
PMT Signal 
Sample DNA Reporter (V @ 546 nm) 
(V @ 514 nm) 
______________________________________ 
1 DNA labeled 
Avidin 6.1297 2.4788 
with linked 
digoxigenin 
Phosphor 
and biotin 
2 DNA labeled 
Silanized 
1.0528 4.4022 
with Phosphor 
digoxigenin 
and biotin 
3 No DNA Avidin 1.6302 3.5779 
linked 
Phosphor 
4 DNA labelled 
Avidin 0.8505 2.8067 
with 
digoxigenin 
and biotin 
5 DNA labelled 
FITC- 1.0484 8.4394 
with Avidin 
digoxigenin 
and biotin 
6 No DNA FITC- 1.0899 3.5779 
Avidin 
______________________________________ 
Phosphor Downconversion Evaluation 
A sample of the (Y.sub.0.86 Yb.sub.0.08 Er.sub.0.06).sub.2 O.sub.2 S 
phosphors were scanned for the presence of a downconverted signal. This 
was accomplished by exciting a sample of the monodisperse phosphors 
described above (4.times.10.sup.-12 M in DMSO) with 1.3 mW of 
monochromatic light at 350 nm with a 16 nm bandwidth for the excitation 
source. Detection was accomplished by scanning this sample from 350 to 800 
nm with a monochomator bandwidth of 8 nm. Scanning was performed in 2 nm 
increments. No downconversion was observed. Moreover, no downconversion 
was seen at the excitation wavelengths cited by Tanke et al. (U.S. Pat. 
No. 5,043,265). Thus, the upconverting phosphors tested are unlike those 
reported in Tanke et al. 
HOMOGENEOUS ASSAYS 
The multiphoton activation process characteristic of upconverting phosphors 
can be exploited to produce assays that require no sample washing steps. 
Such diagnostic assays that do not require the removal of unbound phosphor 
labels from the sample are herein termed homogeneous assays, and can also 
be termed pseudohomogeneous assays. 
Homogeneous Assay Example 1 
One embodiment of a homogeneous assay consists of the use of an 
upconverting phosphor label linked to an appropriate probe (e.g., an 
antibody or DNA). The phosphor-labeled probe specifically binds to a 
target (e.g., antigen or nucleic acid) that is linked to a capturing 
surface. A suitable capture surface can be the tip of a light carrying 
optical fiber (FIG. 23) or the bottom surface of a sample container (FIG. 
24A and 24B). upon incubation of the target-labelled capture surface with 
the phosphor-labelled probe, phosphor particles will accumulate at the 
capture surface as a function of the amount of target present on the 
capturing surface. The target may be linked directly to the capturing 
surface or may be immobilized by interaction with a binding agent (e.g., 
specific antibody reactive with target, polynucleotide that binds target) 
that is itself linked to the capturing surface (such as in a sandwich 
immunoassay, for example). 
Detection of the phosphor bound to the capture surface is effected using an 
excitation light that is focused from a low intensity beam of large 
cross-section to a high intensity beam of small cross-section with the 
focal point of the beam being at or very near the capture surface. 
Focusing of the excitation light is accomplished by transmission through 
optical elements that have a very small focal length, such that the beam 
diverges and becomes less intense, within a short distance of the capture 
surface. 
Since the intensity of the light emitted from the upconverting phosphor 
labels is proportional to the excitation light intensity raised to a power 
of two or greater, phosphors near the focal point of the excitation source 
will emit significantly more light than those remaining in suspension in 
the sample away from the capture surface. Therefore, binding of 
upconverting phosphor linked probes to the capture surface will yield an 
increase in emitted light intensity measured from the sample as a whole or 
as measured from a control sample in which phosphors do not bind to the 
capture surface. Emitted light intensity may be plotted as a function of 
target concentration using for standardization (calibration) a series of 
samples containing predetermined concentrations of target. The emitted 
light intensity from a test sample (unknown concentration of target) can 
be compared to the standard curve thus generated to determine the 
concentration of target. 
Examples of suitable homogeneous assay formats include, but are not limited 
to, immunodiagnostic sandwich assays and antigen and/or antibody surface 
competition assays. 
Homogeneous Assay Example 2 
Another embodiment allows for the accumulation of upconverting phosphor 
linked probes at the detection surface by the application of centrifugal 
or gravitational settling. In this embodiment an upconverting phosphor is 
linked to multiple probes. All the probes must bind to the same target, 
although said binding can be accomplished at different locations (e.g., as 
antibody probes may target different epitopes on a single antigen). The 
multiprobe phosphor can then be used to effect the aggregation of targets 
in solution or suspension in the sample. This aggregation will result in 
the formation of a large insoluble phosphor-probe-target complex that 
precipitates from solution or suspension (FIG. 25). The aggregated complex 
containing phosphors accumulates at a detection surface while 
nonaggregated material remains in solution or suspension. Detection is 
accomplished as described in the above example using a sharply converging 
excitation beam. 
Evaluation of Up-converting Chelates 
Up-conversion has been performed in rare earth chelates and rare earth salt 
solutions. Chelates of erbium and neodymium have been prepared with 
ethylenediaminetetraacetic acid (EDTA) and dipicolinic acid (DPA). The 
erbium chelates were pumped using light near 793.5 nm from a Ti:sapphire 
laser (the excitation scheme of Macfarlane (1989) Appl. Phys. Lett 54: 
2301). This approach produced upconversion but not satisfactorily, which 
we attribute to weak absorption for the first step due to the increase in 
linewidth in the chelate over the low temperature crystal used for the 
up-conversion laser. 
The neodymium chelates were excited with light near 580 nm from a 
Nd:YAG-pumped dye laser (following the excitation scheme of Macfarlane et 
al. (1988) Appl. Phys. Lett 52: 1300). An emission spectrum for the 
emitted up-converted light at 380 nm is shown in FIG. 32. We estimate the 
up- conversion cross section to be 10.sub..box-solid. 
--27&gt;&gt;cm.sub..box-solid. 2&gt;&gt; for this experiment. 
We have also observed up-conversion in thulium acetate hexahydrate and 
holmium chloride hexahydrate in solution following the excitation schemes 
of Allain et al. (1990) Electron. Lett. 26: 166, and Allain et al. (1990) 
Electron. Lett. 26: 261, respectively. The salts were dissolved in heavy 
water, and excitation was performed using a krypton laser. Although the 
up-conversion was weak, the up-conversion should be improved if chelated 
compounds are used instead of dissolved salts. 
Although the present invention has been described in some detail by way of 
illustration for purposes of clarity of understanding, it will be apparent 
that certain changes and modifications may be practiced within the scope 
of the claims.