Automatic recording fluorometer/densitometer

An automatic recording fluorometer/densitometer has an integral viewer for viewing and photographing fluorescent samples which have been applied to a clear, thin-film substrate. Light from an ultraviolet source is on the opposite side of the sample from the viewer when the sample is placed in a viewer holder. When the sample is placed in a recording holder, light from the same source is on the opposite side of the sample from the recorder optics. By having the source on the opposite side from the optics, it is possible to have the source much closer to the sample and to thereby reduce the stray ultraviolet light in the instrument. Another source emits visible light for densitometric analysis of the sample. A movable filter holder can be indexed to different positions for placing an ultraviolet filter or one of the densitometric filters in the optical path between the sources and the recording device.

BACKGROUND OF THE INVENTION 
This invention relates to an automatic recording fluorometer/densitometer. 
Zonal electrophoresis has been successfully used in clinical laboratories. 
Electrophoresis is a separative procedure based on the phenomenon that 
electrically-charged molecules or particles will migrate through a 
solution or gel in response to an externally applied voltage gradient. 
Many biological molecules, especially proteins, carry a net electric 
charge in solution which makes them susceptible to electrophoretic forces. 
In the zonal electrophoresis technique, a small volume of biological fluid 
(e.g., blood serum, cerebrospinal fluid, etc.) is applied at one spot on a 
buffer-saturated membrane or thin-layer medium. When a voltage difference 
is established across the ends of the medium, different molecular species 
in the sample migrate with different velocities, and with time, resolve 
into a series of distinct bands or spots. The desired molecular bands can 
be visualized by direct staining with specific dyes, or in the case of 
certain enzymatic samples, by applying chemical substrates that become 
catalytically converted to colored or fluorescent products. Deviations 
from normal in the intensity or mobility of the bands revealed by a given 
test are associated with biochemical abnormalities that may indicate a 
particular pathology. 
Electrophoresis is often effective in separating nearly identical 
biomolecules, since minor differences in charge and molecular conformation 
may result in noticeably different electrophoretic mobilities. Similar 
electrophoretic procedures can be applied to a wide variety of clinical 
tests, since the specificity of a given test is largely determined by the 
dyes or reagents used in the final processing. The simplicity, rapidity 
and versatility of zonal electrophoresis, as well as its general 
effectiveness, make it an attractive basis for clinical determinations 
that require the separation of biomolecules. 
U.S. Pat. Nos. 3,479,265 and 3,635,808 disclose thin film agarose plates 
which can be used as the electrophoretic medium. The thin film plates of 
these patents are particularly convenient for handling and storage. 
Electrophoresis preparations or samples can be grouped into two categories, 
densitometric and fluorometric. Densitometric (sometimes called 
colorimetric) samples have bands that absorb visible light and are thus 
observable in normal room light. Fluorometric samples absorb ultraviolet 
light and fluoresce, emitting light at visible wavelengths. Thus 
fluorometric samples cannot be seen in normal room light but must be 
excited with ultraviolet light to be observed. 
Qualitative clinical evaluation of electrophoresis samples can be done by 
visual inspection, i.e., gross abnormalities can be detected in this way. 
However, the present state-of-the-art in clinical medicine typically 
requires a more critical quantitative evaluation. 
Quantitative evaluation of electrophoresis preparations requires the 
generation of an optical density profile or fluorescence intensity profile 
of the sample, whichever is appropriate. 
The optical density profile is partitioned into individual peaks each 
representing a band on the electrophoretic separation. Adjacent peaks are 
separated by recognizing and selecting a valley. Integration of the area 
under each individual peak and computation of the peak area as a 
percentage of the total area under the profile represents, for example, 
the distribution of certain proteins in blood serums. Norms for such 
distributions have been established, and deviation from these norms is of 
diagnostic significance. The percentage numbers are sometimes multiplied 
by a "scale factor" so that the results are in units of protein 
concentration or enzyme activity, rather than percent of total. 
One instrument for automatically making analyses of the aforementioned type 
is described in U.S. Pat. No. 3,706,877. 
In addition to automatically recording the concentration distributions of 
the film, it is desirable to directly view the films. Fluorometric 
iso-enzyme samples have bands of iso-enzymes dispersed along the length of 
any given sample which cannot be seen in room light. The sample must be 
shielded from visible light and illuminated with ultraviolet light in 
order to see the iso-enzyme bands. 
Currently available viewers for fluorescing samples usually make use of a 
hand held source of ultraviolet radiation in a darkened room or a sample 
is inserted into a box and viewed through a port with the sample being 
viewed from the same side that it is illuminated. Inherent in this 
arrangement is both reduced intensity and reduced uniformity since bulbs 
must be placed outside the field of view and relatively distant from the 
sample. State of the art fluorescent viewing devices do not provide 
operator protection from exposure to ultraviolet radiation. 
SUMMARY OF THE INVENTION 
In accordance with this invention, a single instrument provides a 
comprehensive quantitative evaluation of clinical electrophoretic samples. 
In accordance with this invention, the optical density and fluorescence 
intensity of a sample are measured and recorded in an instrument which can 
be changed between the fluorometric mode of operation or the densitometric 
mode at different light wavelengths as appropriate to the particular 
sample being evaluated. 
In accordance with this invention, an integral fluorometric viewer makes it 
possible to visually inspect fluorometric samples as a screening operation 
prior to further evaluation. 
In accordance with a further aspect of the invention, fluorometric and 
densitometric light sources are on the opposite side of the sample from 
the recording device and the fluorometric source is located on the 
opposite side of the sample from the viewer. This permits the light 
sources to be quite close to the sample to maximize excitation of the 
sample, while at the same time the sources do not block the optical path 
between the sample and the recorder or the optical path between the sample 
and the viewer. 
In accordance with another important aspect of the invention, good 
shielding of the ultraviolet light is provided. The ultraviolet light 
sources are located close to the sample and on either side of the optical 
axis so that geometric shielding of the ultraviolet light from the 
recording device is possible. 
The integral viewer includes an ultraviolet blocking filter located in the 
bottom opening of the viewer in such a manner that the observer is 
completely shielded from ultraviolet light. Ambient light is excluded from 
the viewer. Because the ultraviolet source is on the opposite side of the 
sample, it can be positioned quite close to the sample for good excitation 
but the observer does not see the source because of a selective 
transmission filter. 
The foregoing and other objects, features and advantages of the invention 
will be better understood from the following more detailed description and 
appended claims.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The instrument of this invention includes a case 11 which encloses the 
fluorometric and densitometric sources and optics. For automatic 
recording, the film sample is inserted in the recording sample holder 12. 
The sample holder slides into the case between the light sources and the 
optics for the recording system. The scanning stage 13 moves in a 
horizontal direction to scan the light across the film. Concurrently, a 
recording pen 14 moves across the chart 15. A light detector measures 
light intensity from the sample, and the recording system responds to the 
output of the detector to move the recording pen 14 orthogonally to the 
scanning motion. This records the intensity of fluorescing or 
densitometric light from the sample. The recording device is more fully 
disclosed and claimed in co-pending application Ser. No. 799,942 filed May 
24, 1977, Amos et al, Recording Analyzer for Electrophoretic Sampler. 
In order to select the mode of measurement, a mode selection knob 16 is 
provided. This knob is set to make either a fluorometric analysis or one 
of three densitometric analyses at wavelengths of 450, 520 and 600 
nanometers. 
An integral viewer includes a shroud 17. The viewer is used to visually 
inspect fluorometric samples as a screening operation prior to further 
evaluation. Shroud 17 is hinged at 18 to the instrument case 11. The 
shroud shields the sample from visible light so it can be illuminated with 
ultraviolet light in order to see the iso-enzyme bands dispersed along the 
length of the sample. The shroud 17 is tilted on its hinge and the sample 
is placed in a viewing sample holder formed between the case and the 
shroud. 
Referring to FIG. 2, the instrument includes an incandescent lamp 19 used 
as a source for making densitometric measurements at different visible 
wavelengths. Lens 20 and fold mirror 21 direct light through one of the 
three densitometric filters 22, 23 or 24 mounted on the filter holder 25 
(FIG. 3) which is moved to place the appropriate filter in the optical 
axis. An aperture stop 26 is in close proximity to the sample 27 and 
determines the optical resolution in densitometry. 
The sample is moved from right to left during its scanning and it absorbs 
light in accordance with the optical density of the sample. Light passing 
through the sample is gathered by the objective lens 28, reflected by the 
mirror 29 and passes through secondary filter 30. Filter 30 is a compound 
filter which transmits only light of the desired band of wavelengths. 
Densitometric light passing through the aperture 26 is imaged on the 
second aperture 31 so all of the densitometry light passes through this 
second aperture. 
An aspheric lens 32 forms an image of the objective lens exit pupil on the 
photo diode detector 33, so that all the light collected by the lens 32 is 
sensed by the detector 33. The output of detector 33 is applied to a 
logarithmic amplifier 34 which produces an electrical signal proportional 
to optical density or absorbence of the sample. This signal is applied to 
the recording device 35 which records absorbence. 
Photo diodes are linear devices. A signal proportional to the density of 
the sample is equal to a constant times the logarithm of the light passing 
through the sample. In order to use the same detector 33 and recording 
device 35 for both densitometric and fluorometric measurements, it is 
necessary to use a logarithmic amplifier 34 in densitometric measurements. 
In the fluorometric mode of operation, the filter holder 25 is moved to 
place the ultraviolet transmission filter 36 in the optical path. Mode 
switch 37 is changed to apply the output of detector 33 to a linear 
amplifier 38. The aperture 26 is changed from an 0.4 .times. 2.5 
millimeter aperture to a 8 .times. 13.5 millimeter aperture. Another 
switch, not shown in FIG. 2, deenergizes the incandescent lamp 19 and 
energizes ultraviolet lamps 39 and 40. 
Light from ultraviolet lamps 39 and 40 passes through the ultraviolet 
transmission filter 36 and through the aperture stop 26 to excite the 
sample 27. The emitted fluorescence is collected by the objective lens 28 
and passes through the secondary filter 30 which blocks any stray 
ultraviolet light. The objective lens 28 forms an image of the excited 
area of the sample on the second aperture 31. This aperture determines the 
optical resolution of the system in the fluorometric mode. The aspheric 
lens 32 forms an image of the objective lens exit pupil on the photo diode 
detector 33, so that all the light collected by the lens 32 is sensed by 
the detector 33. 
The ultraviolet lamps 39 and 40 are located quite close to the sample and 
at 45.degree. from the optical axis. This minimizes the size of the 
excited area of the sample and controls stray ultraviolet excitation 
light. Excitation light which passes through the sample 27 is 
geometrically excluded from entering the objective lens 28. The system 
provides good geometric and spectral discrimination against excitation 
light. In the prior art, ultraviolet excitation lamps are on the same side 
of the sample as the detection optics. In such systems, it is virtually 
impossible to exclude ultraviolet excitation light from the detection 
system. 
The dual imaging system including movable aperture stop 26 gives good 
fluorescence collection efficiency, precise control of optical resolution, 
and allows use of a small area solid state photo diode detector. 
The mode selector is shown in FIGS. 3 and 3A. FIG. 3 shows the mode 
selection mechanism for indexing the various filters and their respective 
apertures precisely over the optical center line 41. Filters 22, 23, 24 
and 36 respectively pass 600 nanometer, 520 nanometer, 450 nanometer and 
fluorometric light. An aperture is provided beneath each filter. The index 
mechanism includes knob 16 which moves lever 42 pivoted at 43. 
FIG. 3A shows a cross-section through the mechanism exposing primary spring 
44, secondary spring 45 and push rod 47. When the knob 16 is depressed, 
the bias on ball 48 is released, allowing it to be moved out of one of the 
detents 49 in the way rod 50. The filter holder 25 slides along way rod 50 
to a new position. 
The purpose of the secondary spring 45 is to maintain slight pressure on 
the ball 48 so that the operator has some feedback "feel" and knows when a 
detent is present. 
Mode selection switches 51 have an actuating arm 52 which is actuated by 
the movement of the holder. When the optical axis passes through the 
fluorometric filter 36, as shown, the actuator 52 is depressed, thereby 
turning on the ultraviolet lamps 39 and 40 and connecting detector 33 to 
the linear amplifier 38 (FIG. 2). 
In any position other than fluorometric, the actuator 52 of the mode switch 
is released, thereby turning off the ultraviolet lamps, turning on 
incandescent lamp 19 and connecting the detector 33 to the logarithmic 
amplifier 34. 
FIGS. 4 and 5 show the integral viewer in more detail. 
An ultraviolet pass filter 53 is mounted in the opening on top of the case. 
An ultraviolet blocking filter 54 is hingedly mounted in the bottom of 
shroud 17. As shown in FIG. 5, the shroud 17 is tilted back and the 
blocking filter 54 is raised on its hinge so that the film sample 57 can 
be placed over the pass filter 53. Hold down strips 55 and 56 cooperate 
with the edges of the pass filter 53 to form a viewing sample holder. 
With the shroud tilted back, a camera rests on the ultraviolet blocking 
filter 54. This enables the operator to take pictures of the samples he 
has viewed. 
An opening 60 (FIG. 4) is used to view the fluorescing sample. The shroud 
of viewer 17 almost totally excludes ambient room light from the area of 
the fluorescing sample 57 which is subject to uniform ultraviolet 
illumination from the lamp 39. The same lamps are used for viewing as for 
recording. The operator is shielded from ultraviolet light by the 
instrument case and by the ultraviolet blocking filter 54 in the bottom of 
the viewer. The viewer of this invention is a marked improvement over the 
prior art in that illumination of the sample is from underneath. This is 
possible because of the selective transmission filters which prevent the 
light source from being visible to the operator. This integral viewer 
provides an operator work station for viewing and photographing samples 
which can then be automatically scanned and fluorometric or densitometric 
measurements can be recorded. 
While a particular embodiment of the invention has been shown and 
described, various modifications will occur to those skilled in the art. 
The appended claims are, therefore, intended to cover all such 
modifications which fall within the true spirit and scope of the 
invention.