Fluorometric method for detecting abnormal tissue using dual long-wavelength excitation

A method and apparatus for in vivo detection of abnormal tissue in patients by irradiating a diagnostic region simultaneously with at least two wavelengths of incident light, and detecting the resulting fluorescence of normal and abnormal tissue. The patient is provided with a photosensitizer which preferentially collects in abnormal tissue, and beams of light--preferably at about 612 and 632.8 nm--are directed to the diagnostic region. The beams of light are chopped at 90 and 135 Hz, respectively. Fluorescent light from the diagnostic region is then detected, and an electronic signal is generated relating to the intensity of the fluorescence. Because of the chopping of the incident beams, the fluorescent light and the resulting electronic signal are also chopped. The electronic signal is provided as input to phase-locked amplifier circuitry, which differentiates between the contribution to the signal resulting from each of the 612 and 632.8 nm incident beams. A difference signal is provided as output to headphones, and the operator of the apparatus is notified of presence of abnormal tissue by changes in pitch of the difference signal. The source for the light may be lasers or an arc lamp, and there may be three or more incident wavelengths used.

BACKGROUND OF THE INVENTION 
Animal tissues contain traces of materials, such as protoporphyrin, which 
fluoresce at a wavelength of 690 nm when excited by visible light. Such 
fluorescence is described, for example, in the article by R. H. Pottier et 
al., "Non-Invasive Technique for Obtaining Fluorescence Excitation and 
Emission Spectra In Vivo," Photochemistry and Photobiology, Vol. 44 pp. 
679-687 (1986). Tissue fluorescence is also discussed in the article by 
William R. Potter and Thomas S. Mang, "Photofrin II Levels By In Vivo 
Photometry," Progress in Clinical and Biological Research, Vol. 170 pp. 
177-186 (1984). The above articles are incorporated herein by reference. 
The fluorescent tumor localizing photosensitizer Photofrin II is retained 
by abnormal tissue such as tumors at a higher level than most surrounding 
normal tissues, and therefore it is diagnostically useful to supply 
Photofrin II to the tissues, and then to illuminate the tissue with light 
to detect by the fluorescent response whether abnormal tissue is present. 
In the therapeutic use of this material (referred to as photodynamic 
therapy, or PDT), large doses of 630 nm light are used both to activate 
the fluorescence of the sensitizer (such as Photofrin II) and to 
selectively destroy the tumor by a photochemical reaction However, the 
fluorescent response of tissues may be created by excitation using 
incident light with wavelengths in the 600 nm region, which is in the 
visible spectrum, and thus there is a problem with stray light causing 
fluorescence which may be interpreted as arising from abnormal tissue. 
Thus, there is a need for a system which can accurately differentiate 
between fluorescence arising from sensitizer in normal tissue and that 
arising from sensitizer in abnormal tissue, especially in vivo. In 
addition, there is a need for distinguishing between fluorescence arising 
from low levels of fluorescent tumor localizers (i.e., sensitizers such as 
Photofrin II) and natural tissue background fluorescence. 
There is especially a need for a fluorometer which can detect abnormal 
cells which are within a mass of tissue, such as within a group of lymph 
nodes, without the need for slicing the tissue open and inspecting each 
sliced segment in a superficial manner, as has been done in the past. 
Thus, it is an object of this invention to provide a method and apparatus 
of fluorometry with the capability of effectively penetrating a mass of 
tissue for purposes of detecting abnormal tissue. 
One characteristic of presently used PDT methods is the need to use 
therapeutic levels of the sensitizer which result in highly photosensitive 
skin for long periods of time, often on the order of four to six weeks. 
This skin sensitivity requires the patient to remain indoors during 
daylight hours after injection until the photosensitivity has decreased. 
Thus, long and high photosensitivity is a significant disadvantage to the 
use of this drug for detection or localization. The need to use high 
levels of the drug is a result of the natural background fluorescence of 
the tissue, which tends to vary in a random fashion from point to point. 
In one system, an imaging device uses 400 nm absorption for superficial 
excitation of bladder tissue. H. Baumgartner et al., "A Fluorescent 
Imaging Device for Endoscopic Detection of Early Stage 
Cancer--Instrumental and Experimental Studies," Photochemistry and 
Photobiology, Vol. 46, No. 5, pp. 759-763 (1987). In this system, tissue 
is first scanned using light in the violet region of the spectrum, and a 
subsequent scan with green or blue light from an argon laser is used to 
excite the tissue background and subtract this contribution to the image. 
There are certain disadvantages to this approach, however, one of which is 
that the tissue excitation by the two wavelengths is done in an 
alternating fashion, such that real-time images of in vivo tissues are not 
achievable, since registration of the image would have to be maintained 
for the two excitation wavelengths. Furthermore, it would be impractical 
to use this type of imaging with light in the 600 nm range because 
scattering of the light by tissue would cause resolution to be very poor. 
However, imaging with wavelengths of light in the 600 nm range is highly 
desirable because of the deep penetration of such wavelengths. There is 
therefore a need for a system for in vivo fluorometry which produces 
real-time images which may utilize longer wavelengths for noninvasive 
examination of tissue to the maximum depth possible, especially for use 
with handheld probes. There is also a need for a system which utilizes 
relatively low levels of sensitizing chemicals such as Photofrin II, so as 
to greatly reduce or eliminate clinically significant photosensitivity. 
It is an object of this invention to provide a method and apparatus for in 
vivo fluorometry which can be implemented in a handheld nonimaging probe 
where sequential tissue excitation is not feasible. 
SUMMARY OF THE INVENTION 
The present invention comprises a method and apparatus, including an in 
vivo fluorometer, employing simultaneous dual long-wavelength excitation 
to cancel tissue background fluorescence by subtraction. The apparatus of 
the invention includes two lasers for providing two beams of incident 
light, one at 612 nm and one at 632.8 nm. The light beams are chopped, 
i.e. periodically interrupted, by a tuning fork chopper, one at 90 Hz and 
the other at 135 Hz, at two other chopping frequencies chosen to exclude 
mutual harmonics. The two beams are combined into one diagnostic beam by 
means of prisms and a lens, and are directed through an optical fiber to a 
diagnostic region of a patient or animal pretreated with Photofrin II or 
some other local tumor photosensitizer. 
Both normal and abnormal tissue will fluoresce as a result of the incident 
beams, and a receive fiber is coupled to the transmission fiber to pick up 
such fluorescence. The transmission and receive fibers are coupled 
together in a fixed geometrical relationship, forming a probe. 
The fluorescent signal is filtered by a 690 (.about.10) nm optical 
interference filter, and is converted to an electronic signal with a 
signal strength related to the intensity of the fluorescence The 
electronic signal is provided as input to each of two tuned amplifier 
circuits, which are designed to filter out the contributions to the 
fluorescent signal from the two incident beams. Thus, one filter 
effectively extracts the contribution to the fluorescence which results 
from the 612 nm beam, and the other extracts the contribution resulting 
from the 632.8 nm beam. An A channel and a B channel are provided in the 
circuitry for carrying the two electronic signals. 
The apparatus is calibrated in advance to ensure that, when no abnormal 
tissue is present, the A channel signal equals the B channel signal. If 
abnormal tissue is present in the patient, the A channel signal will 
increase significantly, due to the fluorescence of the sensitizer in the 
abnormal tissue. A signal (A-B) is generated by subtraction circuitry, and 
is converted to an audio signal with an audio frequency related to the 
magnitude of the difference, and the audio signal is provided as an output 
to headphones for the operator of the apparatus. The operator is thus 
notified of the presence of abnormal tissue by an increase in the 
frequency of the audio signal. 
Circuitry may also be provided to generate a signal (A-B)/B, which is 
independent of the distance from the probe to the diagnostic region, and 
is also independent of other factors which influence the fluorescent 
signal such as attenuation due to a tumor being situated beneath a layer 
of other tissue. The operator may optionally select the (A-B)/B signal for 
input to the headphones, and digital voltmeters are also provided for 
visual display of the A, B, A-B and (A-B)/B signals. 
An oscillator circuit is provided for driving the choppers and for 
providing a phase-lock signal to each of the A and B channels for accurate 
detection of the respective contributions to fluorescence from the two 
incident wavelengths. The phase-lock signal is conditioned by removing 
harmonics and converting it to a sine-wave signal. 
Thus, the apparatus and method of the invention accomplish the needs 
described above, including providing real-time in vivo detection of 
abnormal tissue and avoiding erroneously identifying normal tissue as 
abnormal. Low levels of photosensitizers such as Photofrin II may be used 
without loss of accuracy of the results, and natural tissue background 
fluorescence is precisely subtracted out of the fluorescent signal. Use of 
wavelengths in the 610 and 630 nm range both allows for deep penetration 
of the tissue and takes advantage of an intensity peak for Photofrin II.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As shown in FIG. 1, the apparatus of the invention includes two lasers such 
as HeNe lasers 10 and 20, in front of which are placed choppers 30 and 40. 
The choppers may be model L40 HHD tuning fork choppers with a Type HEA-5A2 
driver, produced by American Time Products division of Frequency Control 
Products, Inc. of Woodside, N.Y. The integrated circuits shown in FIG. 1 
are available from Analog Devices of Norwood, Mass. 
FIG. 1B shows the fluorometer 1, which includes digital voltmeter displays 
2 and 3 and switches 4 and 5, as well as a probe 6, with functions to be 
described below. 
The choppers 30 and 40 shown in FIG. 1 are driven by oscillators 50 and 60, 
respectively. Oscillator drives chopper 30 at 135 Hz, and the oscillator 
60 drives the chopper 40 at 90 Hz. Each chopper blocks the laser beam 
exiting from its associated laser at the rate driven by the oscillator in 
question. Thus, the chopper causes a 135 Hz, 632.8 nm laser beam to reach 
a prism 70, and likewise a 90 Hz, 612 nm laser beam reaches prism 80. The 
612 nm HeNe laser is available from PMS Electrooptics of Boulder, Col., 
and the 632.8 nm HeNe laser is available from Spectra Physics of Mountain 
View, Calif. Instead of the 612 nm laser, another laser having a 
wavelength of approximately 610 nm may be used. 
The beams are combined by a prism 90 acting in conjunction with a 
planoconvex lens 100. The convergent laser beams are fed into an optical 
fiber 110, and the laser light is conducted thereby to a treatment site, 
such as treatment site 120 shown in FIG. 1. 
The subject or patient, such as rat 130, is first given an injection or 
otherwise supplied with a sensitizer such as Photofrin II. Such a 
sensitizer will be preferentially concentrated in the treatment site 120. 
The Photofrin II will fluoresce due to the excitation of the laser light, 
in particular due to the laser light at 632.8 nm. As described below 
relative to FIG. 12, there is a fluorescent peak at an incident wavelength 
of approximately 630 nm. 
As both the tissue background fluorescence (excited by 612 nm and 630 nm) 
and the Photofrin II fluorescence (excited by 632.8 nm) are detected 
simultaneously at 690 nm, a means must be provided for separating the two 
contributions to the 690 nm fluorescence. This is accomplished by the 
choppers 30 and 40, which cause periodic interruptions in the incident 
beams and consequently also in the resulting fluorescence at 690 nm 
wavelength. The apparatus and method for differentiating between the 
contributions to the fluorescent signal by the 612 and 632.8 nm incident 
wavelengths are discussed below relative to fluorescence detection. 
It is an important feature of the invention that the user is enabled to 
simultaneously detect the normal tissue background and the abnormal 
tissue, since this allows the abnormal tissue to be ablated at the same 
time as detection, with a high degree of accuracy, in an in vivo setting. 
There are no image registration problems which are inherent in sequential 
imaging techniques. 
The 90 Hz signal (resulting from the 612 nm excitation) represents the 
tissue background or "B" channel. at 690 nm together with the effects of 
any stray exciting light which may leak through the 690 nm interference 
filter 160 shown in FIG. 1. The half-power band pass of this filter is 
preferably about 10 nm. The 135 Hz signal (resulting from the excitation 
at 632.8 nm) represents the tissue background fluorescence plus the 
Photofrin II fluorescence at 690 nm together with stray exciting light. 
Because the tissue background excitation efficiency is nearly identical for 
the 612 nm and 632.8 nm excitation frequencies, the subtraction of the two 
signals produces (A-B), which accurately represents the Photofrin II 
signal only for all depths of the tissue, as discussed below. That is, the 
two exciting wavelengths are close enough together that they behave nearly 
identically in tissue (have similar scattering and absorption properties) 
and are nearly identical in their leakage through the 690 nm pass filter 
used to eliminate almost all of the exciting light from the detector. 
Thus, it is possible to adjust the amplification of the 690 nm 
fluorescence produced by 612 nm excitation to cancel signal in normal 
tissue not containing the Photofrin II (or containing a low level of 
Photofrin II). 
In practice, the magnitude of the background signal is cancelled, i.e. 
reduced virtually to zero, by adjusting the gain of the "B" channel using 
normal tissue without the sensitizer present, in the calibration technique 
described above. More significantly, the random point to point 
fluctuations can be reduced by a factor of eight in normal tissue with or 
without a low level of Photofrin II. (The factor of eight was determined 
by measuring the fluctuations in both the A and B channels by means of 
voltmeters attached to these channels; the B-channel fluctuations turned 
out only one-eighth as large as the A-channel fluctuations.) 
Fluorescence is picked up through another optical fiber 140, which is 
preferably held directly against the treatment site 120. Light emanating 
from the fiber 140 is collimated by a planoconvex lens 150, and is 
transmitted through an optical band pass filter 160, which may be an 
interference filter centered on 690 nm, with a filtration band of plus or 
minus 10 nm. The band pass filtered diode may be a DFA 6900 produced by 
EG&G Electrooptics and Electronics of Salem, Mass., and the optical fibers 
may be model number HCR-M400T-12 from Ensign Bickford Optics Company of 
Avon, Conn. 
Once the dual-wavelength optical signal is filtered, it is processed by a 
fluorescence detector 170, as shown in FIGS. 1 and 2. The fluorescence 
detector 170 produces a signal relating to the intensity of the input 
signal, and this is then fed into each of two tuned amplifiers 180 and 
190, as shown in FIG. 2. The amplifiers (marked as Q1 and Q3 in FIG. 2) 
are tuned in a standard fashion by the "parallel T" or "twin T" feedback 
method. The twin T network has the property of a high impedance at the 
desired frequency and a low impedance elsewhere. Thus, the amplifiers Q1 
and Q3 have a voltage gain of 100 at 90 Hz (Q1) and 135 Hz (Q3), and their 
gain falls rapidly at other frequencies. The one-half power bandwidth is 
approximately 5 Hz, and thus the "Q" (representing bandwidth/center 
frequency) is about 25, which is fairly close to the highest "Q" for which 
an amplifier can be made unconditionally stable. Circuits with higher "Q" 
are prone to oscillation. 
The gain of the Q1 circuit 180 (and hence the "Q" value) is limited by the 
feedback provided by the 100K and 4K voltage divider. The gain of the Q3 
circuit 190 is achieved in an equivalent manner by using the single 3.1 
megohm resistor shown in FIG. 2. The Q3 circuit 190 requires fewer 
components. Depending upon the open loop gain of Q1 and Q3, the 4K and 3.1 
megohm resistors may require some adjustment up or down to achieve a gain 
of 100. The value of R/2 (shown as a variable resistor at the bottom of 
circuit 180) is adjusted to approximately 33K, and the value of R'/2 
(shown as a variable resistor at the bottom of circuit 190) is adjusted to 
approximately 22K, and these adjustments are fine-tuned to bring the pin 6 
and pin 3 signals of Q1 and Q3 into phase. 
The buffer stages Q2 and Q4 are used to prevent loading the amplifiers Q1 
and Q3, and to allow the fine-tuning of the gain of each channel. 
Channel A is calibrated to give a consistent signal using a standard made 
by dissolving Kiton Red dye in ethylene glycol. The fluorescence of Kiton 
Red is extremely stable, and therefore this dye is particularly suitable 
for laser use. Channel B is adjusted to null the value of A-B when normal 
tissue or unsensitized tissue is fluoresced. The gain of these stages is 
typically 3 to 10. 
The calibration technique for nulling the signal in channel B due to 
background fluorescence is as follows. The "A" channel gain is adjusted 
using the calibration control. A Kiton Red dye fluorescence standard is 
used for calibration. The Kiton Red (produced by Exciton Inc. of Dayton, 
Ohio) is dissolved in ethylene glycol (0.0615 g/l). The probe 6 is cleaned 
using distilled water and lens tissue and held perpendicular to the side 
of a 1 cm square cuvette containing the fluorescence standard. Care must 
be taken to avoid letting fingers get in the way, since finger tips are 
fluorescent. The calibration control is adjusted to give 0.100 volts on 
the "A" channel digital voltmeter 240. 
The null control is then adjusted (A-B) to read -4.200 volts. This will 
give an approximately zero (A-B) reading on normal human skin without 
Photofrin II, that is, it has been empirically determined that a reading 
of -4.200 volts should be used for calibration when the fluorescence 
standard is Kiton Red, but of course other fluorescence standards might be 
used for calibration. Moreover, it may be desirable to use another setting 
for calibration if a very different tissue is used in a particular study 
or if it is necessary to compensate for a high background level of the 
sensitizer; although in the latter case, one should probably consider the 
use of lower doses of sensitizer. The calibration is, of course, carried 
out before the diagnosis begins. 
In order to differentiate between the A and B signals, accomplish this, the 
signal from the silicon photodiode (produced by the light passing through 
the filter 160) is fed into two tuned amplifiers and then into two 
detectors, each of which is phase locked to the appropriate chopper drive 
signal, as discussed in greater detail below. This technique rejects 
everything but the fundamental signals of 90 Hz in one channel and 135 Hz 
in the other channel. Because the two frequencies are at odd half 
multiples of one another, the phase-locked detection completely rejects 
any cross interference between channels at the fundamental and at all the 
harmonics as well. Other frequencies may be used, preferably chosen to 
exclude common harmonics. 
The A and B signals, once processed by the circuitry 180 and 190, are fed 
into the lock-in amplifier circuits 200 and 210, which are identical in 
structure and are represented jointly in FIG. 3. As shown in FIGS. 1 and 
3, a phase reference signal is provided to each of the circuits 200 and 
210. Thus, the oscillator 50 has an output which is fed through a signal 
conditioner 220 and ultimately as a phase reference signal to the 
amplifier circuit 200. Similarly, the oscillator 60 has an output signal 
which is fed through the signal conditioner 230, and ultimately as a phase 
reference signal into the amplifier circuit 210. 
In the signal conditioners 220 and 230, the input phase I reference signals 
from the oscillators 50 and 60, respectively, are square waves. To remove 
the harmonic content, each square wave is attenuated and then amplified by 
a stage identical to the tuned stage Q1 or Q3. The conditioned signal 
should be as close to a pure sign wave as possible for satisfactory phase 
shifting. In particular, since the degree of phase shift is frequency 
dependent, it is undesirable to have harmonics in the phase reference 
signal. 
The amplifier circuitry 200 and 210 is similar to that found at Volume I, 
Section 6, page 65 of Analog Devices 1984 Databook. This circuit produces 
a full-wave detection or rectification and filtration of the signal 
provided at pin 16 shown in FIG. 3. The detection is in phase with the 
reference signal applied to pin 9 shown in FIG. 3. 
The pin 9 signal selects either an inverting or a non-inverting 
amplification (with a gain of 1) of the pin 16 signal. When the signal on 
pin 9 changes polarity, the selected amplifier changes. The phase 
adjustment is used to compensate for phase shift between the mechanical 
motion of the chopper and the referenced driving voltage. 
The AD515 stage shown in FIG. 3 is used to filter the output of the lock-in 
amplifier. There is a trade-off between noise and band pass, and for noise 
in the range of 3-5 mv, the 0.1 second time constant produced by the 10 
K/10 .mu.fd combination is adequate. 
FIG. 4 shows a subtraction circuit utilizing a differential amplifier 
AD521KD to subtract the filtered output of the two lock-in amplifiers. The 
signal which results (A-B) is the 690 nm fluorescence produced by the 
632.8 nm excitation, minus the 690 nm fluorescence produced by the 612 nm 
excitation. 
FIG. 5 shows a division circuit which ratios the difference signal (A-B) to 
the tissue signal (B), which produces a signal (A-B)/B which is 
independent of the distance the probe 6 may be from the treatment site 
120. If the user sets switch 5 (in FIG. 1) such that the (A-B)/B option is 
chosen, the audio tone will still increase with increase in A (and hence 
with the presence of abnormal tissue), but the signal will be compensated 
for accidental variations in the distance from the probe to the diagnostic 
region, such as tissue 120, by the dividing process. Dividing the 
difference signal by the background also makes the result independent of 
the strength of the tissue excitation and of the efficiency of the 
collection of the fluorescence of the tissue. This quantity, (A-B)/B, is 
most influenced by changes in the amount of Photofrin II present. It also 
tends to be independent of spurious changes in the fluorescence signal 
caused by changes in the optical attenuation properties of the tissue. 
This is true because the 612 and 632.8 nm wavelengths are close together 
in a region where the optical properties of tissue do not change radically 
from point to point in a different fashion for each of these two 
wavelengths. 
Changes in the optical properties or the efficiency of the tissue 
fluorescence at 690 nm as the probe is moved about would also be perfectly 
compensated for by (A-B)/B, because such changes would appear as a 
constant multiplier of both the numerator and denominator of this 
expression and thus cancel. For instance, if a tumor is buried beneath one 
to several millimeters of normal tissue, the 690 fluorescence due to the 
632.8 nm beam will be attenuated; however, the 690 fluorescence due to the 
612 nm incident beam will be attenuated by an identical factor, and thus 
the attenuation factor will cause the values of A and B to decrease. Since 
this attenuation factor appears as a multiplier of both A and B, it 
cancels out in the expression (A-B)/B. 
Shown in the lower portion of FIG. 1 are two digital voltmeters 240 and 
250, with connections to each of the outputs A, A-B, (A-B)/B and B shown 
at the upper right of FIG. 1. Each of the commonly-named connector points 
are connected to one another as shown in FIG. 1. Thus, for example, when 
voltmeter 250 has its switch 5 connected to connector point B, as shown in 
FIG. 1, it receives the output B from the amplifier circuit 210. 
A voltage-to-frequency converter 260 is connected to the output of the 
voltmeter 250, and headphones 270 are attached to the converter 260. If 
the switch 5 of the voltmeter 250 is connected to the (A-B) or (A-B)/B 
connector points, then as the value of A increase, the frequency supplied 
to the headphones 260 will also increase. Typically, a clicking noise will 
be heard in the headphones 270, and a faster clicking, ultimately becoming 
an apparently continuous and rising pitch, will be heard as the value of A 
increases. Since the value of A depends upon the 135 Hz signal, an 
increase in A and hence an increase in the frequency of the signal in the 
headphones 270, indicates the presence of a greater amount of the 
sensitizer (such as Photofrin II), which in turn indicates the presence of 
a tumor. Thus, the operator the device may utilize the fiber optics to 
scan a treatment site, and can detect the presence of abnormal tissue 
simply by listening to the headphones 270. 
The voltmeter 240 provides a visual readout analogous to the audio signal 
of the voltmeter 250, and thus provides a precisely quantified visual 
signal through the operator. Likewise, the voltmeter 250 may be provided 
with a visual readout or dial, and thus two visual readouts (which is one 
for A and one for B) may be provided at the same time as the audio signal 
over the headphones 270. 
The signal conditioners 220 and 230 may be of the design shown in FIG. 6, 
and will be identical except for the value of R, which is chosen to 
produce the 135 Hz and 90 Hz phase reference sign waves respectively. 
FIG. 7 shows a design for the probe 6 for use in connection with the in 
vivo fluorometer of the present invention, which will accomplish linear 
scanning from purely rotary motion. This can be done with an array of 
fibers. The fibers would be arranged in a straight line at the tissue end 
of the probe 6 and in a circle at the instrument end of the probe cable. 
The order of the fibers would need to be preserved (that is, no fibers can 
be allowed to cross others before the fibers are fastened together side by 
side at each end). Scanning of the circular end with a laser beam focused 
to a point is readily done by a round window with a 10.degree. wedge angle 
(that is, with nonparallel faces). This wedge would deflect the beam as it 
passed through it and could be rotated about an axis through its center 
and perpendicular to one of the planes of the wedge. The center of the 
fiber circle would also pass through the extension of this axis of 
rotation and the plane of the fiber circle would also be perpendicular to 
the rotation axis. If the wedge angle, the fiber circle diameter and the 
lens focal length are appropriately chosen, then the focused spot will 
sweep around the circle formed by the flat polished ends of the fibers. As 
this circle of fiber ends is a linear array at the tissue end of the 
fibers, the effect is to translate to pure rotary motion into a linear 
scan with essentially no time lag between the end of one scan and the 
beginning of the next. 
This principle could also be used to scan the image of an aperture in front 
of a filtered detector diode across a second circle of receiver fibers. If 
the aperture were of the correct size, then all the light from each fiber 
in turn could be scanned across the detector. Thus, two rows of parallel 
fibers could be arranged in transmitter and receiver pairs and be 
sequentially activated to scan a line across the tissue. 
FIGS. 8 and 9 show a probe design utilizing a zirconium oxide sphere as a 
focusing lens for the transmission fiber 110. The receive fiber 140 may by 
provided in multiple, such that six receive fibers 140 are actually 
utilized. The utilization of the spherical lens 280 allows for uniform 
illumination over a circular area, and the equal spacing of the fibers 140 
picks up fluorescence from tissue around the periphery of the illuminated 
circular area. 
The surface probe of FIGS. 8 and 9 is especially useful for the examination 
of large areas (e.g., breast cancer metastatic to the chest wall after 
mastectomy). Although it is referred to as a "surface" probe, this probe 
will actually produce an exciting light field with a larger illuminated 
area wherein the light is more slowly attenuated with depth. 
The exciting light is conveyed to the probe by the transmitting fiber. The 
surface of the end of this fiber is imaged by the 1 mm diameter zirconium 
oxide sphere onto the surface of the tissue. The advantages of using such 
a sphere as a lens are several, including that sealing and handling 
problems during construction are greatly reduced. The sphere is held by a 
0.001 inch undersize press fit into the brass body of the probe. This is 
only possible because of the great mechanical strength of the zirconium 
oxide sphere, which is available from Precomp Inc. of Great Neck, N.Y. 
Another advantage is that it provides a highly uniform illumination of the 
surface. 
The six receive fibers which contact the tissue provide a system which 
compensates for the lower power density of the exciting light, is highly 
symmetrical (and thus insensitive to probe rotation) and most sensitive to 
fluorescent targets located beneath the center of the field. The probe is 
thus capable of accurate localization of deep tumors while at the same 
time covering an area which is big enough to allow a rapid examination of 
large surfaces. 
FIG. 10 shows another configuration of the probe 6 for use in connection 
with the present invention, including a receive fiber and a transmission 
fiber, wherein the receive fiber 140 is 1 cm longer than the transmission 
fiber 110. In use, the fiber 140 is placed directly against the area to be 
illuminated, as shown (although not in scale) in FIG. 1 relative to the 
rat 130, and the transmission fiber 110 illuminates the area adjacent to 
the point of contact between the fiber 140 and the treatment site. Since 
fluorescence takes place in a region all around the area illuminated by 
the fiber 110, such fluorescence will take place immediately beneath the 
point of contact between the fiber 140 and the treatment site 120. 
The transmission fiber 110 is preferably attached to the receive fiber 140 
at a distance of approximately 1 cm from the tissue. The fibers are fixed 
together in parallel fashion by a 1 cm length of heat shrink tubing. The 
transmission fiber conducts the chopped 612 and 632.8 nm light to the 
tissue. This results is an illuminated circular field approximately 3 mm 
in diameter, with a minimally-sized probe. This probe is especially useful 
for examining lymph nodes of 1-10 mm and for use through the biopsy 
channels of fiber optic endoscopes. 
The method and apparatus of the present invention utilize a relatively long 
wavelength (632.8 nm) incident light for excitation of the 690 nm 
fluorescence of the Photofrin II. This is done to allow the maximum depth 
of noninvasive examination of the tissue. Tissue is more transparent to 
light in the red region of the spectrum, as reflected in FIG. 11, which 
shows the attenuation coefficient--that is, the rate at which incident 
light intensity falls off with increasing distance into the tissue--as a 
function of wavelength of the incident light. Although the sensitizer 
absorbs more light in the 400 nm region (see FIG. 12), the tissue 
absorption makes the 630 nm excitation more efficient for depths greater 
than about one millimeter. 
FIG. 12 shows the intensity of fluorescence of Photofrin II as a function 
of the wavelength of the incident light, i.e. the light that excites the 
fluorescence. (The wavelength of the detected fluorescent light is on the 
order of 690 nm.) A very high peak appears at about 400 nm, indicating a 
high fluorescent response to incident light of this wavelength. The graph 
of FIG. 12 shows fluorescence peak intensities of decreasing size as the 
frequency of the incident light goes up, including a peak at about 630 nm. 
For purposes of penetrating tissue, longer wavelengths are, as described 
above relative to FIG. 11, more effective. There is thus a trade-off 
between the intensity of the fluorescent response and the penetrating 
characteristics of the incident light. 
It has been found that the fluorescent response of normal tissue to 
approximately 630 nm incident light is very nearly the same as the 
fluorescent response of normal tissue to approximately 612 nm incident 
light. The present invention utilizes this characteristic by providing 
incident light of both 612 and 630 nm, in a manner to be described below. 
The present method is based upon the in vivo absorption band shape of 
Photofrin II in the 630 nm region and is greatly facilitated by the 
availability of HeNe lasers to produce the required exciting wavelengths. 
In FIG. 13, the in vivo biological action spectrum (which corresponds to 
the in vivo absorption spectrum) is shown for a human patient. In FIG. 14, 
the in vivo fluorescence excitation spectrum is shown for an amelanotic 
melanoma in a rat tumor system. Both of these figures demonstrate that the 
fluorescence intensity peak produced by the approximately 630 nm incident 
light for Photofrin II in tissue. It will be noted that there is an 
approximately 5 nm shift between the fluorescence peak in FIG. 12 (which 
appears at about 625 nm incident wavelength) and those of FIGS. 13 and 14, 
which appear closer to 630 nm wavelength. This is a result of conducting 
the tests of FIGS. 13 and 14 in vivo, and the shift in the peak may be a 
result of binding of the sensitizer to proteins or other substances. 
FIG. 13 was produced using an argon ion pumped dye laser as a tunable 
excitation source for the 690 nm fluorescence of the tissue. The spectrum 
was collected in a noninvasive fashion using fiber optic probes touching 
the surface of the patient's skin during PDT treatment. The effects of the 
tissue background are evident in the failure of the fluorescence to return 
to baseline away from 630 nm (e.g. at 612 nm). Also apparent is the rise 
in the baseline as the exciting wavelength increases. This is due to the 
leakage of the exciting light through the 690 nm pass filter over the 
detector (a silicon photodiode). The use of the 612 nm light as background 
cancellation is advantageous because it is as close to the 630 nm peak as 
possible. That is, the 612 nm wavelength is chosen to be as close as 
possible to the beginning of the rise of the 630 nm peak on the left side 
thereof as shown in the graphs of FIGS. 12 and 13, without actually being 
on the portion with the increasing slope. It has been found that the 
response of normal tissue to 612 nm excitation is very similar to the 
response to 630 nm excitation, whereas abnormal tissue treated with 
Photofrin II responds quite differently to these two wavelengths, as is 
evident from the 630 nm peak of FIG. 13. 
Choice of the 612 nm excitation wavelength for use in conjunction with the 
630 nm wavelength therefore results in the best selectivity for the 
Photofrin II absorption and in the most nearly identical scattering and 
absorption behavior with depth in the tissue. The two exciting wavelengths 
will behave in a similar fashion as they penetrate tissue so that the 
cancellation of background will be accurate at all depths. 
In an alternative embodiment, a third wavelength--at, for example, 638 nm, 
which is adjacent the 630 nm peak on the right side of FIG. 13--could be 
used to produce an average baseline for background correction. For this 
purpose a broad spectrum light source such as an arc lamp could be used in 
conjunction with a diffraction grating three exit slits to provide three 
different wavelengths for excitation. As an alternative to the diffraction 
grating, portions of the emission from the arc lamp can be directed 
through three interference filters to provide three different excitation 
wavelengths. Other photosensitizers absorbing at even longer wavelengths 
might also be utilized. 
In general, lasers may be preferred as light sources because the beams are 
spectrally clean and stable with respect to wavelength, and tend to be 
more reliable and rugged than arc lamp sources. Also, lasers generally 
provide higher power than arc lamps, which makes it easier to detect the 
fluorescence signals, and masks noise in the detectors. However, when 
three or more light sources are used, a single arc lamp may become more 
practical than several lasers. 
Typically, there will be random independent fluctuations in the output of 
the two HeNe lasers, on the order of approximately 1-2% of total power. 
Although these power output shifts are small, they can become significant 
in a subtracted application. In one embodiment, compensation for 
fluctuation in the HeNe power is accomplished by a voltage-controlled 
amplifier stage. The output of the HeNe would be sampled using a glass 
plate at 45.degree. to the beam axis, thus directing a few percent of the 
power to a photodiode with tuned amplifiers and lock-in detection 
identical to the fluorescence detection. The signals from these two 
lock-ins would be used to control the gain of an additional stage of 
amplification in each of the fluorescence detector channels. Thus, 
variations in the excitation which are linearly reflected in the 
fluorescence signal would be canceled by corresponding opposite variations 
in the amplification of the fluorescence signal and the noise of the 
system would be reduced and its sensitivity increased. 
An apparatus for this purpose is shown in FIG. 1A, which is an alternative 
configuration to the apparatus of FIG. 1. The circuitry of FIG. 1A 
includes a tuned amplifier 290 which may be essentially identical to the 
amplifier 170, except that the resistor in the former is variable and has 
a range to approximately 10.sup.7 Ohms. This acts in conjunction with the 
two conventional voltage-controlled amplifiers 300 and 310 to regulate the 
amplification of the A and B channel signals, respectively, in response to 
variations in the output power of the lasers which result, for example, 
from variations in the line voltage supplied to the lasers. 
The amplifiers 300 and 310 may include the LM13600N amplifier produced by 
National Semiconductor, which is a dual operational transconductance 
amplifier. One design for the amplifier if FIGS. 300 and 310 is shown in 
FIG. 1C. 
The amplifier 290 includes a photodiode 320 which detects a sample of the 
beam incident upon a partially reflecting mirror 330. The signals from the 
amplifier 290 ultimately reach the amplifiers 300 and 310, which 
compensate for laser output power fluctuations. 
STEP-BY-STEP PROCEDURE FOR USE OF THE FLUOROMETER 
1. Power up the apparatus--plug in machine and turn it on. 
2. Allow a ten-minute warm up. 
3. Clean the probe 6 with lens tissue (soft) and distilled water. 
4. Calibrate according to the above. 
5. Sterilize the probe 6 by soaking it for ten minutes in Cidex (1% 
gluteraldehyde solution). Then rinse the probe in sterile water. 
6. Touch the probe 6 to the tissue of the diagnostic region. Keep finger 
tips away from the exciting light, since finger tips are fluorescent, even 
through latex gloves. 
7. Notice the reading of (A-B)--or (A-B)/B--and use the rising pitch of the 
headphone sound as a quick guide to interesting areas of higher 
fluorescence as the probe is moved over the tissue. 
8. Use the large area probe (such as in FIG. 8) to examine large areas of 
skin or other large surfaces. 
9. Examining small (1-10 nm) nodes during surgery is done with the "node 
probe" (see FIG. 10). This probe is small enough to use through a fiber 
optic endoscope by passing through the biopsy channel. In this application 
it may not always be possible to hold the probe perpendicular to the 
surface and the distance between the tissue and transmitting fiber may be 
effectively varied. It is for this situation that the (A-B)/B option 
discussed above was included to compensate for the decreased efficiency 
that such geometric problems produce in both transmission into the tissue 
and reception back from the tissue. 
10. Areas of high fluorescence may be removed and examined histologically 
to define the pathology of the tissue. It is helpful to use the 
fluorometer to guide the excision as the device responds to microscopic 
amounts of tumor. Similarly, the pathological examination should be 
exhaustive to avoid missing one or two microscopic nests of tumor cells 
which can be detected by the fluorometer. 
Variations on the foregoing may be made and still utilize the teachings of 
this invention. For instance, other methods may be utilized for imparting 
characteristics to the incident light beams so that they may later be 
differentiated in place of chopping the beams into different frequencies. 
Other embodiments may be arrived at without departing from the spirit and 
scope of the invention.