CA1086093A - Fluorometric system, method and test article - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A fluorometric system to determine the kind and amount of substances derived from a biological fluid (e.g., serum or urine) or tissue in which the substances to be detected (e.g., antigen, antibody, hormone or enzyme) are coated onto a substrate surface in fluorescent form. Multiple coating areas of different samples may be employed. The fluorometric system includes a source of filtered light to excite fluorescence, optical systems for conducting the excitation light to such coating, and optical systems for receiving emitted fluoroescence and for detecting the same. The system efficiency and optical characteristics disclosed avoid photo-bleaching; limit fading; and are especially adapted to provide accurate surface reading fluorometry.

Description

Back~round of the Invention This invention relates to fluorometric systems for the detection of sample substances deri~ed from biological fluid or tissue tagged with fluorochromes and to fluorometers adapted for more accurate measurements of sur~ace mounted fluorescent samples. It is particularly useful in the detec-tion of hormones, enzymes, drugs and other substances.
Most infectious diseases of bacterial or viral nature produce antibodies in the blood serum of the subject.
This provides a degree of immunity against future assaults by the identical infectious agent or antigen. One method for detecting the presence of a particular antigen is to add to it a specific antibody which binds to the antigen.
I the antibody has been previously tagged with a radio-active element (RIA technique) or a fluorescent dye, which does not interfere with its immunological proper~ies, the coupled sample can be detected by an appropriate detector;
and, in the case of the fluorescent additive, can be at best semiquantitatively measured, as is done in cases in the prior art on a microscope slide for visual inspectionO

There are many reasons why RIA is not completely , satisfactory. For example, in the presence of small quan-tities of antigen, only few counts per second can be de-tected. Since the "noise" of the system is proportional to the square root of the signal count, large errors in accuracy are made at low signal levels. Furthermore, radio-isotopes have a limited shelf life due to half-life decay, and require special licensing, handling and disposal.

!
. - 2 -,~ "
.~. ., .

~66)~3 ."

,.,, . ~ .
i~ Testing which relies on fluorescence tagging techniques, as heretofore known, has been qualitative, or at best, semiquantitative as an assay. In the area o~ the largest diagnostic use of fluorometry ~i.e., immunofluor-escence microscopy in which samples are typically mounted on a microscope stage and illuminated with an exciting ; wavelength) the fluorescence is observed through the stage ~ with appropTiate filters interposed to select the wave-length to ~e observed. Typically, the resultant observation is recorded by a laboratory technician as a comparative degree of fluorescence, for example 0, ~1, +2, +3, or +4 b~ comparison to known reference concentrations. In some lnstances, where blood titre or concentration of antibodies is the desired unknown, the technician prepares a number of slides; on each is a different concentration of the test ;~ material. Thus, the technician may estimate a +4 reaction in the microscope when the blood serum or the bacteria `
broth medium was diluted 1:4 in distilled water, or 1:16, or 1:128, etc. It would be of great advantage to medi~al ; 20 and clinical authorities if a fluorometer could automati-cally and quantitatively read titre or concentration quick-, ` ly and nccurately, without the necessity of making serial dllutlons.
- In liquid scanning fluorometers, a cuvette usu-ally holds a liquid containing the substance to be analyzed " and through which excitation light is passed and fluor-escence observed in ~ right angle configuration. It has been found that these systems are unsuitable for the present applications primarily because liquid systems and cuvettes themselves, apart from the sample being investi-';-; ':

r 36~93 .

gated all contribute very substantial background fluores-- cence, so that, unless a very high degree of careful chemical separation is utilized together with extremely well-controlled materials selected to have low fluorescence in the wavelengths of interest, such systems are unsuitable.
Attempts to adapt these instruments for surface measurements have not been particularly successful. Such systems are typically inefficient and have not provided for the dis-crete handling of individual samples. In general, past 10 fluorometers and RIA systems have been unduly sensitive to background and non-sample oriented signals. There is, ; therefore, a need for a new and improved fluorometric system.
Summary of the Invention and Objects In general it is an object of the present inven-tion to provide a fluorometric system and method which will overcome the above limitations and disadvantages. Another object of the invention is to provide a fluorometric system of the above character which is quantitative, which is ; easily calibrated and which is arranged to efficiently operate wlthin acceptable limits relating to photo-bleaching and fading. Another object of the invention is to provide an improved optical fluorometer particularly adapted to read ; samples disposed on a carrier having and presenting a free sample surface thereon, and in which the optical system receives 1uorometric data solely from the sample area of sa:ld carrier. Another object of the invention is to `~ provide both fiber-optic and lens optical fluorometric systems for carrying out the invention.

; 4 _ ;~

. ~ .

:'~

... , . . , . - .

The fluorometrlc s~stem of the present invention measures a sample substance coated over a surface on a solid substrate. It includes a source of light filtered to selectively excite fluorescence in the sample and light-conducting means for conducting light ~rom the source to the sample. An emitted light detector captures and deter-mines the intensity of fluorescence Rmitted rom the sample substance by converting the fluorescent light intensit~ to an electrical signal by a photodetector. Fluorescence is conve~ed from the sample area to the photodetector by suit-able light collection optics terminating proximally adjacent the sample and distally adjacent to the photodetector! The optical parameters of the various elements interposed between the sample and photodetector define a light path which is constructed and arranged in accordance with this invention to prevent excessive loss of fluorescence (i.e., cumulative loss is held less than 95%). Thus, the disclosed light collection optics (such as fiber optical cables or lenses) must be constructed with limited gap distances at each end and with a large collection aperture in order to enable efficiency of this level to be obtained over the emission band peak.
~ n additional and related criteria is that the eoregoing must be achieved within the constraint that the lnput light intensity shall be held to a value less than causes photo-bleaching or photo-disa~sociation resulting in a fading rate of one tl%~ percent per minute. B~
achieving specifications that meet the foregoing, a very useful and accurate instrument is disclosed.

.
:

. . . . . .
.

6~3 .:, -- The coated substrate to be viewed in the fluoro-meter may comprise a single sample coating on a body. Alter-natively, a body adapted to enable detection and determination of more than one sample substance may include multiple spaced coating areas (e.g., bands) of different substances. A single ; coated area may include different substances in random dis-persion tagged with different fluorochromes.
In one embodiment, the fluorometric system includes ~- a hranched fiber optical cable for conducting light from the source to the sample and for conducting emitted fluorescent - light from the sample to the detector. One branch conducts light from the source to the sample and the other conducts the fluorescent light to the detector. These branches meet in a common fiber bundle terminating at the sample end. In this manner, the area of coincident excitation and emission is maximized at extremely close gap distances and optical efficiency is obtained.
-:
Another advantageous fiber optical system includes at least two fiber optical cables or conducting the emitted

- 2~ llght to the detector and means for alternating th0 inpu-t to the detector between the cahles. This system can read at least two coated areas on a single substrate without move-ment of the substrate as in a comparision between a standard ; ~uantity o~ sample and one or more unknown samples. Similar-~', ly, both the light conducting means to excite fluorescence and to receive fluorescence comprise branched optical cables for transmitting multiple wavelengths of light to the sample and receiving different fluorescent signals.

.' ,: .

. . i . :

8166~93 , .. .

- A third preferred embodiment constructed in ac-cordance with the present invention uses lenses systems intersecting a removable stage forming an analysis test ... .
station. The stage is adapted to receive a member having a surface forming a carrier to a fluorometrically active sample substance adhered thereto and presen~ing a free sur-face for examination. The member carrying the sample is constructed and arranged for insertion and removal from the stage independently thereof and is adapted to form a light-tight arrangement with the stage so as to exclude background light from the optical systems. In addition, the stage and :~ carrier member together with the associated optical compon-ents and housing serve also to form an enclosure for the sample contained therein which enclosure avoids the circula-tion of ambient atmosphere ~i.e., usually air). In this ,j - preferred embodiment the surface portion is arranged hori-,; zontally and includes a slight depression so that the sample ~` may be disposed thereon in liquid form and so remain to . .~
present a free liquid surface for exposure and examination during the analysis. By means of the foregoing, the sample ::
is maintained quiescent in movement and in a stable, low evaporative enviro~nent for analysis. As in the previous embodiment, the optical systems maintain the excitation light intensity and collection efficiencies within the limits ~` deEined by the present invention, as will be more clear from i the detailed description herein.
.~ ~
` In each oE the foregoing embodiments filters are `~ utillzed for defining the input and excitation wavelength band and for establishing a band width of sensitivity in the 3a detection system. For optimum performance, these filters .
, : . ' ., . , : :
~ . - ' ~., ' . :

have been ~ound to be very critical and detailed speci~ication for their selection will be ~iven.
. According to the broadest aspect of the lnvention, ` there is provided, in apparatus or analyzing a sample by fluoro-metric techniques, a carrier member having a surface portion : adapted for receiving said sample and includin~ means for support-ing said sample thereon as an exposed layer presenting a surface ~or analysis, test stage means for supporting said member at an analysis location in said apparatus, said carrier member and .- lO said stage means being constructed and arranged so that said member is removably insertable into said stage means, illumi-- nation means including a source of excitation radiation and means forming a first optical system intersecting said analysis location for delivering said radiation to the surface thereat, optical fluorescence collection means including a photo-detector and means forming a second optical system .intersecting said analysis location for receiving the fluorescence emitted . from the exposed layer thereat, emission filter means associated :
-` with said fluorescence collection means for restricting the ; 20 sensitivity thereof to a frequency band overlapping the emission spectra o~ said fluorometrically active substance and being substantially non-responsive to the band of excitation xadia-tion, means juxtaposed with said excitation filter means for forming a beam splitter for removing a portion of the light therefrom - Por developin~ a reference channel in parallel with the illumination means, means for alternating said illumination means and said reference means during mutually exclusive periods of -time, sensing means for developing a signal indicative of sample illumination on-times when said photodetector is activated by sample fluorescence and for developing a second signal indicative of the period when said reference means is detected, ~-~ a demultiplexer response to said signal for alternately deliver-~ `'~'' .

~ . . . . . . .
. ~ . . . . .

~6~93 - ing outputs indicative of said sample signal or said reference ; signal, a sample signal processor for receiving said sample signal, a reference signal processor for receiving said reference signal, said processors serving to develop signal outputs, the strength of which is proportional to the amount of light signal received at said photodetector during said sample and reference .. periods, respectively, a ratio circuit for obtaining the ratio S/R where S equals sample signal and R equals said reference ~ signal, and display means responsive to said ratio circuit for P 10 displaying the value of said ratio.
These and other objects and features of the present invention will become apparent from the following detailed description of the preferred embodiments when taken in conjunc-- tion with the accompanying drawings, of which:
BRIEF DESCRIPTION OF THE DRAWINGS
- Figure 1 is a top, schematic view of one embodiment of a fluorometric system constructed in accordance with the present invention using fiber bundle optical systems; .
Fiyure 2 is a cross-sectional view, in elevation, taken along the lines 2-2 of Figure l;
. Figure 3 is a schematic diagram of the photo-detector and associated circuitry for use in the embodiment of Figure l;
. Figure 4, on the first sheet of drawings, is a ` schematic view of another embodiment of the fluorometric system ~- constructed in accordance with the present invention utilizing a sample carrier in the form of a cylinder;
Figure 5 is a schematic view of an alternate sample member configuration constructed in accordance with the present invention;
` 30 Figure 6 is a schematic view of another alternate sample member configuration constructed in accordance with the present invention;

:
;` .

36~93 - Figure 7 is a graph relating concentration of fluore-scent material in sample (C) and intensity of detected signal ::~ (Io) as a function of efficiency of the optical collec-tion system and as a function of input light intensity;
Figure 8 is a schematic, isometric view of another preferred embodiment of fluorometer constructed in accordance : with the present invention, shown with external portions in ;~ phantom lines;
Figure 9 is a detailed optical diagram of the fluoro-- 10 meter of Figure 8;
Figure 10 is a perspective view of a test stage assembly including sample holder constructed in accordance with the present invention and particularly adapted for use in the ~; fluorometer of Figure 8;
.~ Figure 11 is a cross-sectional view, in elevation, of the test stage assembly of Figure 10;
. Figure 12 is a cross-sectional view taken along the lines 12-12 of Figure 11;
Figure 13 is a top plan view of the sample holder of Figure 10;
Figure 14 is a graph depicting performance curves of ~` filters constructed in accordance with the present invention for use in the embodiment of Figures 8-13;
Figure 15, on the third sheet of drawings, is a bloc]c diagram of an electronic circuit for use in the fluorometer of Figures 8-14;
Figures 16 and 17 are schematic views similar to that of Figure 1 of modified forms of fiber optic fluorometers con-structed in accordance with the present invention; and ~ 30 Figure 18 is a cross-sectional view of a fiber-optic ~ cable taken along the lines 18-18 of Figure 17. :

: -9a-~" :

~, , . . . , ~ .

166~3 - DESCRIPTION OF THE PREFERRED EMBODIMENTS
:, Tbe present in~ention relates to a fluorometric system and method to quantitatively detect and measure a fluore-scent sample substance coated as a layer on a substra~e. As de-' flned herein, the term "fluorescent sample substance" is one which -`- includes a material derived from either a biological fluid ortissue and which, alone or in combination with other materials in a solid layer form, emits fluorescence upon excitation with a ~"~:
~-~ selected wavelength of light in a solid layer form. Common fluor-escent sample substances include autofluorogenic material derived from a biological fluid (e.g., tetracycline), materials derived from such fluids tagged with fluorochrome before or after isola-tlon, materials derived from such fluids linked in the layer with homologous fluorochrome-tagged materials (e.g., antigen or anti-~ body, one of which has been tagged with a fluorochrome). The present description will make particular reference to the last ~; named substance.
Referring now to the embodiment of the invention as ~- shown in FlGURES 1 and 2, there is provided a member 10 having a n surEace portion adapted for forming a carrier for a fluorometric substance which is adhered thereto as a surface coating. In the embodiment shown, this member is in the form of a ball which is prepnred, by way of example, in the following manner.
The ball may be about 10 mm in diameter and made of plastic, e.g. nylon, and bears a dried film or coating 11 of an antibody relatecl to the antigen to be determined, e.g. to Australian antlgen. Since coating of all balls will be done at substantially the same temperature (37C), and for substantially the same incuba-tion period C30 minutes), each ball will have substantially the same - 30 amount of antibody on it, which is important for quantitative re-sults. The ball is typicall~ on the order of 5 - 20 mm in diameter .~

~, .

-6~3 so that an area at least one s~uare mm is viewed by the fluorometer. The ball is placed into a cuvette 12, in this instance of 12 mm inner diameter. It is preferred that fluorescence measurements are made in accordance with the present invention by viewing the surface -to which the fluorometrically active substance is adhered by optical systems which are very closely positioned with respect to that surface so as to maintain a pre-determined level of optical efficiency. However, if fluore-scence is to be detected and measured with the ball in the cuvette, as shown in Figure 1, the cuvette must be formed of a material, e.g. glass, which is non-fluorescing at the wave-length to be measured.
Referring now particularly to Figure 2, an example is now given of the preparation of the ball, it being understood that this description is specific for the sake of giving a detailed background on one procedure for such preparation. ~ `
However, it is to be understood that it is given solely by way o~ example and that many other procedures will be found useful and in accordance with the present invention. Examples of such procedures are given in U.S. Patent application Serial No.
553,582, now U.S. Patent 3,992,631, in greater detail in con-j~mction with many other illustrations and examples of the use oi the fluorometric system set forth herein, the content of which application is not deemed necessary for understanding the invention as presented and claimed herein, and, accordingly, -` will not be repeated.
; The ball is placed in a cuvette 12. One ml. of serum 13 Erom a patient or subject is added to cover the ' -11-. .

.

::;
;`
- ball and the cuvette is gentl~ rocked for 5 minutes to obtain room temperature ~ incubation. Australian antigen 14, if present, binds to the coating 11 of -~ the antibody on the-ball. A cap 16 having a hole 17 is placed on the open end 18 of the cuvette 12 and the cuvette 12 is inverted, permi~ting the serum ; to run out. A small second hole 20 to permit passage of air is also provided in the cap 16. Suitably, the cuvette 12 has a rounded or generally hemisphe-rical inner surface 21 at its base 22, whereby the ball lO is held in posi-tion and does not move or roll around during the fluorescence test.

After incubation with the subject serum, the cuvette is rinsed out, e.g. w1th aqueous phosphate buffer or distilled water, which is then also ` allowed to pour out of hole 17, the ball 10 remaining in the cuvette and anti-body solution tagged with a substance which fluoresces under ultraviolet i light. Such a fluorescent tag or label substance can be, e.g. sodium fluor-escein isothiocyan~te or other suitable substance. Ilowever, sodium fluor-escein isothiocyanate, with excitation at 460 nanometers and emission at 520 nanometers, is advantageous. The material in the cuvette 12 is again incu-~ated as described above, the liquid poured off and the cuvette and ball rlnsed as 6efore. The ball now bears the antigen and attached fluorescent substance 23 where Australian antigen is present. The cuvette 12 and/or mem-ber 10 are now ready for insertion into the fluorometer system.
Referring now to FIGURES l and 3 in the fluorometer system, the member 10 and/or cuvette 12 are so placed that a fiber optical cable 24 con-' ducts ultraviolet or visible light from a light source 27, which can be any desired source which includes the excitation wavelength. The light then pas-- ses through a gelatin filter 28, which ensures that only light of the excit-;Ing wavelength and excluding the emission wavelength reaches the carrier sur-: ~acc on member lO to excite fluorescence of the coupled substance 23. A

second fiber optic cable 25 is disposed preferably at a small angle, less than 30, from the cable 24; and the emitted fluorescence passes through a gelatin filter 30, which ensures that only emitted fluorescence reaches photo-.`"`' .
~ - 12 -.-```~' .

.

36~3 . '.

: detector ~e.g., a photomultiplier) and associated circuitr~ 29 via the fiber optic ca~le 25. The photodetector 29 converts the intensity of the emitted fluorescent light into an electronic signal. This signal is passed through an electronic filter 31 to a processor 31a ~which converts the AC signal to ; a DC signal, e.g. through a peak-to-peak detector and linearizes the relation-ship between fluorescent light intensity and DC voltage, as by a four-step diode linearizer), an amplifier 32, and an analog-to-digital converter 33 and then is displayed on a digital panel meter 34 calibrated directly in titre.
In the embodiment shown in FIGURE 4, a nylon cylinder 35 is em-ployed as a carrier member 10 instead of the ball shape. The upper portion of the cylinder 35 is coated with a standard fluorescent coating 36, i.e. of the same fluorescent substance as is used to tag the antibody coating 38 of tho lower portion of the cylinder 35 and has a known titre as measured on the detector device which is employed in the test or assay; in this instance, the fluorometer described herein. A blank space 37 is left around the surface of c~linder 35, separating the upper and lower coating, 36 and 38, respectively.
The lower coating 38 contains, for example, streptococcal fluorescent-tagged antibody, being prepared in the same manner as described above with respect to the member 10, except that only the lower portion is immersed in the body ~ of li~uid serum to determine if any of the suspected antigen or antibody is present in the serum being tested. In this embodimen~, the ultraviolet or visible light source 27~ the fiber optical cable 24, and the filter 28 are a~ain provided. ~wo fiber optic cables 39 and 47 are provided with respec-~lvc Eilters 3~a and 47a. One such cable 39 conducts fluorescent light from thc stnndard fluorescent coating to the photomultiplier tube 29, and the other s such cable 47 conducts emitted fluorescence from the lower coating 38 to the ; photomultiplier tube 29. A chopper wheel 46 operated by a motor 45 rotates ;.~ and alternates the flow of light from each coating 36 and 38 to the tube 29.
ln this manner, a direct comparison is obtained between the standard reference ~ 30 and the test portions.

:~' .-.
_ 13 -.

36~
, .

In the embodiment of FIGURE 5, the carrier member 10 comprises a paddle-shaped body 40 having a handle 41 at one end extending out of the ; cuvette 12, a stem 42, and a wide, flat head 43 at the other end, the head 43 bearing a coating ~4 o~ sample. In this embodiment~ the two fiber optic cables 24 ~for excitation light) and 25 (for emitted fluorescent light) are parallel to each other, or at an angle o~ 0 with respect to each other.
- Conveniently, the two cables 24 and 25 can also be arranged as a coaxial - cable. The other elements of the device and system are as previously de-scribed and shown.
Another embodiment of multiple test sample carrier body is shown in FIGURE 6. Here, the body is a cube 50 at the end of a handle 51. The tube ; 50 can present four faces, two faces 52 and 53 being visible. Each face has . n different sample. Four different fluorescent tags can be provided, and the Eluorometer may have a filter wheel with four selected wavelength regions to isolate energy going to the photodetector 29.
The following description of FIGURES 7 - 14 relates to a commercial-1~ developed fluorometer developed in accordance with the present invention.
~` Before proceeding further, it will be helpful to consider certain limitations and restraints which are placed upon the fluorometer of the present invention ~0 ln order to achieve the required performance levels.
In general, fluorescence from a sample may be expressed as follows:
Where IF is fluorescent- intensity; IX is excitation intensity; ~ is quantum efficiency; and E, B are geometric factors F X~( ) f ~o~ small concentration of C on a surface e = l-EBC
then IF X~

Let K = ~EB, a physical and geometrical constant for the substance and * 30 s~s~em, then . ,~ - 1~ -:, :

~1) F X
Define IX = EiIs and Io = EoIF where Is is source intensi~y; ID is intensitv at detector; Ei is light transmissive e~ficiency from source to sample; and Eo is light transmissive e~iciency from sample to detector, then C2) ID = KEiEOIsC
Fluorescence fading occurs at high intensities :
IF~t) = KIxCe kt ~here k = koIX and k is a rate constant for decomposition then D(t) i O s For small values of t ~short times) -koEi s = l-k EiIst then t3) ID(t) = KEiEOIsC(l-koEiEst) .. For a practical limit, fading should be less than 1% per minute and this value is given predominatly in that desirable laboratory procedure accomplish-; ment time in one minute and that the largest amount of fading that is accept-able is of the order of 1% per minute. Given these constraints, the follow--; in~ e~uations hold: ..
~, , : o~s9KEiEoIsc = KEiEOIsc-E ~EiEO s) , . .
~o nnd ~- o~OlEiEoIs = Eo(EiEQIS) `s O . 01 = koEiIs ~ t4) ,', I ~ 0.01 :~ s ko i , Substituting ~4) into (2) t5) I~ = O.OlEo .' ko . .

The variables of Equation 5 are illustrated in the graph of FIGURE 7 wherein the line 100 represents the one (1%~ percent fading rate at 100% ef-ficiency ~Eo = 1.0). Above and to the left of thls line, high powers of in-put l-ight intenslty are required and are found to cause photo~bleaching and fading at an unacceptable level and, therefore, cannot be used in this inven-tion.
With the optical systems disclosed herein~ it is possible to a-chieve efficiencies of the order of 5%, the 5% line being indicated at 101 for illustration. The claimed operating domain 103 of ~he present invention lies between lines 100 and 101. As shown, this domain extends down to the limit where the signal and background become comparable in strength ~S/N =
2.0 being indicated at 102) at least to which satisfactory performance is provided by the present invention. Below about 5% efficiency, it becomes im-~ possible to detect low concentrations of sample. It is also seen that the ,~ i fading rate limit and efficiency requirement are mutually interdependent and must both be satisfied. Accordingly, the use of the optical systems dis-:
. closed herein achieves an efficiency of greater than 5% in the collection and detection systems and the input light intensities are held to values such that the fading rate is less than one ~1%) percent per minute.
~n Referring now more particularly to FIGURE 8, there is shown such a lens type fluorometer, the overall outward appearance of which is shown in :~
the phantom lines. The optical system of the fluorometer is developed within -~ an opaque block 104 o~ plastic, as for example Delrin, selected because of ; ~ts dimensional and thermal stability. The block is machined to accept the . ~ .
- various components as shown in the drawings. The details of the machining ~ are not believed essential to understanding the invention and, accordingly, ~;
. thGy have been omitted for the sake of clarity.

`~` Referring simultaneously to FIGURES 8 and 9, the system generally consists of thPee major optical systems: an illumination optical system 105 for supplying excitation light to the sample; a collection ~ptical system 106 , ~
. .:

1~)8~
.
for receiving fluorescent output from the sample; and a reference level op-tical system 107 fGr periodically establishing and checking zero and preset ` intenslty levels.
The illumination optical system 105 consists of lamp 110, condens-ing lens 111, excitation filter 112, chopper 113 and its lens system ll~a and ; 114b, as drivcn by an electric synchronous motor 115 and focusing lens 116 ; for imaging excitation light onto a test stage 150 including sample 118.
The collection optical sys~em 106 consists of a collecting lens 120, emission filter 121, and photodetector lens 123 which images the sample member surface onto a photodetector 124.
The reference level optical system 107 consists of a beam-splitter 130, a turning mirror 131, chopper 113 and associated reference lens system 133, a diffusing screen 134, a portion of which is developed by lens 135 and relnserted at beam splitter 136 into the collection optical system 106. Rota-; tion of the chopper 113 causes light to pass alternately through the illumina-- tlon optical system 105 or through the reference optical system 107. Thus, . . .
the output at the detector is an alternating signal during one period of which the intensity of the excitation source is measured while the other ; period measures the fluorescent output.
- 20 Many of the optical components shown and described herein can be selected from a wide variety of available designs, but the following have been found to be particularly satisfactory for use in the present invention.
By way of example, the lens ll~a, 114b, 133a, 133b, 120 and 135 may be select-cd :Erom the aspheric type produced as standard products oE Mells Griot, pro-~uct S~rlal No. 01 LAG 005. Alternatively, these same lenses may be aspheric lensos having a 2~ mm diameter and an 18 mm focal length produced under the designation as an aspheric condensing lens, stock No. 17.1050 and available from Rolyn Optical Co. of Arcadia, California. Lens 116 is plano-convex with a 25 mm diameter and 50 mm focal length; lenses 111 and 123 are plano-convex lenses with a 25 mm diameter and a 25 mm focal length. Lens 120 is located approximately 0.60 inch from the sample surface.

:

~ IGURE 9 contains ray tracing lines thereon which serve to illus-trate the functions of the lenses and optical components. Thus, lens 111 collimates the light from the light source and passes it in parallel rays through the first filter at 112 after which the beam splitter 130 divides off a certain portion of the light to be delivered to the reference optical sys-tem 107. The remainder passes to the chopper lens pair 114 in the excitation optical system 105. The first lens of this pair 114a focuses the light down to a small spot so that the cut-off and cut-on times and general level of in-tensity of light exiting from the chopper is well-defined in value over an appropriate period of time. The second lens ll~b of that pair collimates the light for delivery to a focusing lens 116 which focuses an image of the fila-~ mont down into a small focused spot on a central area of the sample carrier at 118.
The output from the sample is collected by plano-aspheric lens 120 over a wide solid angle and delivered in parallel rays through the emission , filter 121, through a beam insertion device 136, and thence focused by de-tector lens 123 onto the active element of the photodetector 124.
The portion of the beam which is rem~ved for reference purposes ~s `~ turned b~ turning mirror 131 to follow a path parallel to and alongside the - ~0 o~cltation path. This light is passed through lens pair 133 which also rings the light source to a focus in the plane of the chopper so as to also provide ~or rapid turn-on, turn-of~, and well-defined value o~ intensit~ when ~he raference beam ls on. The reference beam is then passed in parallel rays to a di~fusing screen which can consist of a film of polyester, such as that made by nuPont tType A), having a dull matte finish on the side facing the detectlon system 106. The matte finlsh serves as a light scattering function and thus converts the film into a secandary or reference source of high uniform-ity. ~ portion of the central area of the film is taken by lens 135 and the rays made parallel for being passed to insertion mirror ~par~ial) 136 lying in

3~ the collection stream path between the emission filter 121 and the photode-tector focusing lens 123.

.

86(~3 Re~erring now more particularly to ~IGURES 10 - 13, the sample car-rler stage 150 is shown ln detail. This stage is removable in its entirety to facilitate cleaning. As will be noted from FIGURES 10 - 12, the stage is inserted into a cylindrical passagewa~ in block 104 at a lowermost position such that the carrier 118 containing the sample thereon faces upwardly and in a substantially horizontal plane within the assembly as a whole. By making this provision, together with certain structural features of the sample car-rier, it is possible to insert and measure a sample having a subtantially free liquid containing surface while simultaneously maintaining that surface free of changes during analysis ~such as by evaporation). The stage is rota-tionally oriented for insertion into the optical block 104 by viture of an ` elongate axial slot 151 formed along one side of the stage and adapted to accommodate a locating pin 152 mounted in the optical block. A similar locat-ing pin 152 projecting in the elongate slot 151 indicates when the stage has been brought to a fully inserted position within the block.
The sample carrier 118 is mounted on a removable sample carrier r_ member in the form of a spatula 154 (shown inserted in FIGURES 10 and 11 and separatel~ in FIGURE 13) consisting of an end portion 155 having a substanti-all~ planar surface 156 and an area of slight depression 157 folmed at that end o~ the member on which carrier 118 is disposed to form a support for a film or caating of sample. The spatula 154 fur~her consists of an elongate blade 158 having a registry projection 159 thereon and terminates at its other end - ln a paddle 160 suitable for being easily gripped by the operator. The spatu-ln is easil~ removed and inserted into the stage, the latter normally remain-; ing in place within the instrument. Thus, an elongate stop in the form of slot 162 is provided within the stage for receiving the carrier, together ~itll an upwardl~ extending groove 163 which accommodates projection 159.
When fully home, the parts appear as shown in FIGURE 11. It will be further noted that the outward extremity of the mounting recess 162 (at 164) is pro-v~ded with a converging wedge-shaped configuration so that when the spatula .'" - 19 -:` :

.

9 ~6~93 .
is fUll~ 5eated, the end 166 thereof is urged downwardly against the floor - o~ slot 162 lnto precisel~ positioned contact within the stage. With the utilization of plast~c parts for many of the components, it is found prefer-able that these components be restrained laterally and in every other di~
; mension so that an~ slight warpage of the plastic parts o~ which the carrier mem6er is made is compensated for, the member being urged into exact position against the bottom of slot 162 within the analysis stage.
Consideration of the optical diagram with respect to the focusing of light onto the stage and to the carrier spa~ula and the collection of llght therefrom as shown in FIGURE 9 will explain the purpose for the re-lieved portions 168, 169 provided by the removed portions of the end of stage 150 and laterally located adjacent the sample 118. Once positioned, m0mber 15~ is maintained in position by virtue oE urging contact made by a resilient-ly mounted setting member, inc.luding a spring-loaded ball 170 carried in the body 104 of the optical s~stem and adapted to engage and urge the back in-cllned surface of projection 159 to urge the carrier member inwardly against ~, slot 162 for precise positioning. In addition, ball 170 falls behind that back surface and assumes a position serving together with the projection it-sel~ to block light from passlng through the channel 162. Such light is capable, lf not blocked, of causing non-dark background readings.
An aperture 172 is provided through the stage and passes light :~rom a small light-emitting diode 17~ to a small light detector 173. This aperture is closed by the passing oE the member 15~ as the same is pushed in-to thc unit and when closed provides a "ready" signal for the associated olectronlcs. An additional aperture 176 extends Erom the region immediately ~elo~ the sample carrying end of member 15~ and in general alignment with the excitation light beam so that a photodetector 178 positioned immediately be-hind thls second aperture indicates when the sample is actually in position and blocklng llght from the beam.
The carrier stage 150, even though removable together with its in-, ` :

3L~ 93 dependently removable sample carrier member 154, nevertheless when assembled, forms b~th a light-tight enclosure of quiescent air within the optical as-sembly. The former eliminates background light; the latter retards evapora-tion from an aqueous film over the free sample surface at 118 during the measurement perlod. The latter generally quiescent enclosure is de~ined and formed by the carrler member 154 itself which closes aperture 176, by the stage and its close fitting relationship with the adjacent block, and by ~ lenses 116 and 128, both of which are sealed into contact within bores sup--~ porting the respective excitation and collection optical systems.
In constructing the fluorometer in accordance with the present in-- vention, it is important that the emission ~ilter design be carefully select-ed, and that the excitation filter design compliment that of the emission ~ilter design. In the following discussion, excitation and emission filters will be disclosed which are of substantially identical ~although compliment-,~ ary) structure and which are produced by interference layering techniques in accordance with applicants' specifications and with particular reference to application in the present invention. The examples will be given in connec-t~on with filters designed for use with fluoroscein dye which has an absorp-; tion frequency band at approximately 480 nm ~blue) and an emission frequency band at approxlmately 530 nm ~green). This dye is widely used because of its hi~h quantum efficiency, i.e. about 80 - 90% of the input radiation that is absorbed is re-emitted.
Referring now to FIGURE 14 there is shown a series of curves which illustrate the absorption and emission bands 180, 181 as well as the contours o-~ the chnracteristics of the filters which are constructed in accordance ~ith the present invention. In general, it is desired to obtain a compromise ~ wherein the maximum available input radiation is applied to the sample while obtainlng the maximum output fluorescence emission without overlap or cross-talk between the illumination and collection channels. Since the absorption g~ 30 band 180 of fluorescence and the emission band 181 overlap each other, there ~ .

are certain techniques which must be used ~o obtain optimum performance. In general, the present filters are characterized as follows: a combination pass band, plus cut-on (or cut-off), filters developed b~ interference coatings laid upon a single substate. B~ doing S03 it is possible to obtain a high transmission filter having ~er~ high rejection of the adjacent channel.
Thus, the emission collection filter 121 includes a three-cavity pass band type having a wavelength of highes~ transmission a~ 540 nm and half-width of 16 nm for 50% transmission. This is combined with a cut-on filter having a through transmission of 80% at 550 nm. The pass band curve is shown at 182 while the cut-on curve of this filter is shown at 183 in FIGURE 14 while the combination filter taken together is shown as curve 184. The illu-. .
mination filter 112 is similarly constructed with a pass band curve having acenter wavelength of 475 nm with half-band width of 16 nm, combined with a cut-off filter having its 80% transmission at 465 nm and incorporated with ;I the pass band filter on a single substrate to form a combination -filter of very satisfactory performance. The excitation pass band curve is shown at 185, the cut-off curve is shown at 186 and the combination illumination filter at curve 187. The follo~ing are the specific specifications of interference .~ filters which were made to applicants' specifications and which are available from Ditric Optics, Inc. of Marlboro, Massachusetts.
Illumination Interference Filter CWL: 475 nm ~ 3 nm HBW: 12 to 17 nm 50%
BLOCKING: ~ 4 O.D. outside of passband to 1200nm ~ 6 O.D. from 520 nm to 540 nm ~:, . ~ . . . .. .

. ~ :

a3 .
Collection ~Emission) Interference Filter CWL: 540 nm + 3 nm HBW: 12 to 17 nm ;- %T: ~ 50%
- sLocKING: ~ 4 ~.D. outside of passband to 1200 nm ~ 6 O.D. from 460 nm to 490 nm It will be noted that the crossband rejection is 6 0.D. or .0001%
crosstalk between the channels.

The foregoing filters are generally manufactured¦by a technique known in in~erference filter manufacture as a three-cavity technique and may, of course, employ more cavities as ~he need may require. In general, i~ is ~ound that the foregoing design in displacing the spectra maxima of the fil-ters slightly awa~ from each other and slightly off of the response character-istics of the dye, nevertheless provides very satisfactory performance. In addition, the use of cut-on and cut-of filters on the substrate with maximum transmissions of about 80% yields overall transmission of the desired band of the order of 40 - 50%.
.. Referrlng now to FIGURE 15 there is shown electronic control cir-cuitry which processes the output fluorometer of FIGURE 8 for providing an out-~- put reading therefrom.
In general J the control circuitry serves to time demultiplex the r'-~
~' output of the photo-multiplier tube by suitable circuitry in demultiplexer -. 1~0 dri~en by a synchronizing pulse from a light emitting diode/detector pair 192 posi~ioned across the path of the chopper 113 to derive a sample channel signnl and a reference channel signal, khe respective ones of which are pro-~ cessed ln a sample channel and reference channel respectively. The channels f,' ~ include AC ampli~iers 193aJ 193b, demodulators 194a, 194b, and integrators ;. .
- 195a, 195b for de~eloping DC signals S and R, the magnitude of which is pro-.~ portional to the magnitude of signal strength as seen by the photodetector ;~ 3~ 124 when vie~ing the respective sample or reference channel. The DC signal ~, .
~; - 23 -~,~,.; .
~, .
~"

'. :' ' - ' ~ ,' , ' ' :''`' '. `
;, . ~ . .. . . . . ....

~ 6~93 "'' outputs are passed through respective DC amplifiers 195a, 195b for isolation and applied to a ratio circuit, the output S/R of which is displayed on a digital display unit 198, and also is displayed digitally on ~he instrument's front panel at 198 shown in ~IGURE 8. By using this ratio technique, the stability of the unit is increased and electronic, photomultiplier and illu-mination lamp output drift is minimi~ed.
FIGURE 16 shows a modified form of fluorometer 260 of the fiber ` optic type in which a single branched fiber optic cable 261 replaces the two separate cables 224 and 225. A single-bundle portion 262 of ~he cable 261 leads to and away from a solid base 263 having a fluorescent surface 264. One branch 265 of the cable 261 transmits light from a lamp 266 or other light source and suitable ("blue") filter 267 to the fluorescent surface 264. A
sccond branch 270 of the same cable 261 conducts the emitted fluorescence . from the surface 264 to a suitable ~"green") filter 271 and thence through a lens 272 to a solid state or photomultiplier type of detector 273. Operation is basically the same as in FIGURE 1 with readily apparent differences.
FIGURE 17 shows another modified form of fluorometer of the fiber optic type 280 in which branches 265 and 270 of fiber optic cable 261 re-placed with branch fiber optic cables 281, 282, 283, and 284, respectively.
~ A slngle-bundled portion 286 of the cable leads to and away from a single base 287 having a fluoroescent surface 288. Branches 281 and 282 transmit light from lamps 289 and 290, respectively, or other light sources, to fluor- `
-~ oscont sur~aces 288~ Branches 283 and 284 of the same cable 286 conduct the emitted fluorescent from the surface 288 through suitable lenses 291, 292, respectlvely, to solid state or photomultiplier type of detector 293 and 294, j respectively~
; One method for employing the device of FIGURE 17 which is highly advantageous is to view surface 288 which includes a pluralit~ of biological-~ ly derived substances in random dispersion. Each of the substances is tagged with a ~luorochrome which emits fluorescence responsive to a different wave-. : .:
~'' ;
':~

: . ': ' ,. . - . .

-- ~08Ei;(J~3 . . .

length o~ llght. Thus, lamps 28~ and 290 emlt the different fluorescence exciting wavelengths while the multiple fluorescence is received simultane-ously by detectors 293 and 294 through light conducting branches 283 and 284, respectively. The multiple 1uorochrome tagged substances in random dis-persion may also be read using the single branched fiber optical cable of FIGURE 16. In this instance, a single lamp or other lamp source replaces -lamps 289 and 290 and a plurality of filters are employed to provide the pro-per wavelengths to excite the respective fluorochromes in the samples. Simi-larly~ the light-conducting cables 283 and 284 may be replaced with a single cable and detectors 293 and 29~ may be replaced with a single detector so long as the wavelengths to which the detector is responsive is synchronized to the selected fluorochrome to be excited.
Referring to FIGURE 18, a cross-sectional view of the common fiber bundle 286 of the branched cable 280 is illustrated schematically in which the fibers of the various branches are enlarged for viewing clarity. Such fibers are schematically represented by a solid circle, an open circle, a circle containing "x" and a circle containing "y". It is apparent that the four different types of fibers in this particular arrangement are randomly dispersed. It may be desirable to accomplish a specific optical effect to .
~ arrange them schematically as in concentric circles, not shown, or to use fibers of different diameters. An important feature of the present fluoro-metric system is the maximi~ation of fluorescent light which is received from `~ the sample in accordance with the general criteria discussed in connection wlth ~IGURE 7. This ls particularly important when the fluorescent substance is present at very low concentrations and illumination is held to low values in order to limit fading. In the fiber optic systems of FIGURES 16 and 17, ~ this ob~ective is accomplished by avoiding gap distances in the light path o between the fluorescent substance and the means for converting the light in~
tensity into an electrical signal for quantitative measurement. With a fiber optic cable conducting light from the light input end adjacent the sample to .

60~

the conversion means, such gaps include the distance between the light input . end and the sample substance and any distance between the optical cable and -,:
conversion means.
It has been set forth for average fluorescence intensity, at a mini-mum, the cumulative fluorescence loss across the above light path from sample to detector should not be greater than 95% o~ the fluorescence available for transmission along that path~ Such losses do not include losses due to view-ing only a portion of a fluorescent sample surface. To take this into ac-count, such loss is related only ~o ~he fluorescent light emitted from the sample within an area defined by the ligh~ input end perimeter projected onto the sample surface. Although limiting the loss in fluorescence to 95% across the total light path is a significant improvement over conventional fluoro-meters, it is preferred to limit such loss to below 50 to 90%. At such loss levels, even minute quantities of sample may be detected and determined quan-titatively.
The above considerations deal primarily with fiber optic cables and light pipes and the importance of their close proximity, as expressed by gap distance, to surfaces from which they receive and to which th y deliver light. Light conducting systems may also contain such components as lenses to collect and focus light, mirrors to reflect and redirect it, and apertures :, through which light passes after dispersion. When components such as these receive light from a surface that is radiating it into a hemisphere, the amount of such light they capture is approximately proportional to that por-tion of pi steradians defined by the circumference of the area they project on the hemispherlcal surface generated by a radius equal to the gap distance bet~een the light emitting surface and the component receiving it.
Based upon the above relationship, the circumference which permits loss of no greater than 95% of emitted fluorescence corresponds to one that ~ill generate a solid angle no less than approximately 0.3 steradian. The ` 30 solid angle of non-circular cross-section is defined as one ge ~ated by an ~ 26 -:' ' ' : ' ' ,, .. """ '' :" ''. , equivalent circular area.
A study of the fluorometric systems of the present invention illus- -trate the percentage loss can be directly related to the ratio of the gap dis-tance between the sample and the effective diame~er of the light input end of the emitted light collection optical system. The term "effective diameter"
; means either the diameter of a light input end of circular cross-section or the equivalent diameter of a non-circular cross-section. This latter term may be approximated by reference to the formula:

area: ~d2 ;
The effective diameter, d', of non-circular cross-section is defined as ; 4 x area . Re~erence to the relationship of gap distance to effective diam-~ eter is based upon the approximate relationship that intensity of fluorescence : is inversely proportional to the square of the distance from the fluorescent .. substance. This approximation does not take into account an increase in cap-ture accomplished by minimizing the angle of reflectance, i.e. the angle ~` between the light conducted to the fluorescent substance to excite fluores-~;; cence and that received by the light input end of the detector means. It has been faund that this value is not as significant as the gap distance. It is ;
apparent that the effective diameter of the light input end is significant since an increase in the area of that surface causes a corresponding increase in light captured.
Using the above calculations, a gap distance adjacent the sample ~hich permits loss of no greater than approximately 95% of emitted fluor-t escence corresponds to a ratio of gap distance to effective diameter of the ~-~ light input end of no greater than about 5:1. Similar calculations may be ~nde to determine the theoretical ratio of other fluorescent loss percentages.
lt should be understood that this ratio is only an approximation. The same ~ formula applies to other gap distances in the light path such as between the - fiber optical cable and the portion of the detector which converts the light to an electrical signal and between any lenses and mirrors which may be em-: ` ,.

~"

: .

,.:

; ployed in the l~ght path.
The branched fiber op~ical system of FIGURES 10 - 17 is particular-ly e~fective in reducing to a minimum the gap distance which can be obtained to minimize loss of emitted fluoresccnce. This is ~ased upon the principle that the only area o~ the fluorescent substance which can be received by the detector is ~here the light transmitted to the substance for exciting fluor-escence coincides with the vie~ing area o~ the light input end of the detector.
This can be accomplished with separate fiber optical cables as in FIGURE 1 until a gap distance is reduced to relatively small values. As this reduct}on occurs, the area of coincidence of totally separate light exciting and light emltting cables continuously reduces. It is apparent that this may be a limit-ing ~actor on the gap distance and consequently may cause excessive fluor-escence loss for a sample substance in extremely small quantities. On the other hand, the use of branched cables each including a plurality of light transmitting fibers which terminate in a common fiber bundle at the light input end enable the fluorometer to be disposed extremely close to the fluor-, escent sample without lack of coincidence. The only limit on this is when ;~ the gap distance approaches zero at which point the fine fibers of the fiber bundle act like independent cables.
2n The common ~iber bundle is particularly effective in embodiments such as multiple branching of ~IGURE 17. Cables with separate light input and output ends or each of the branches of cable 280 would require a fairly substantial gap distance to assure a sufficient area of coincidence.
Another technique to avoid loss of fluorescence is to maintain the gnp 6etNOOn the s~lple coating and light input end of the detector free of solid medium which prevents transmission of excessive quantities of fluor-escent light. It has been found that glass or certain plastics (e.g., poly-styrene) at moderate thicknesses of less than 0.05 inch causes a loss of fluorescence less than 30%. Although it is preferable to avoid the inter-position of such a solid medium, such losses are acceptable if necessary or 6~3~3 convenient to the system. ~or example, in the embodiment schematically il-lustrated in ~IGURE 1, it may be convenient to employ a thin-walled cuvette to retain a coated su~strate of a spherical shape. If so, the cuvette should he formed of a material which does not cause the loss of the fluorescence in excess o~ 3~.
- The above description makes reference to ~iber optlcal cables as one pre~erred light conducting means. It should ~e understood that other , optical conduits such as light pipes may also be employed in those instances where the distance does not require the use of common fiber optical bundles.

.s~ .

`, ~

!
.`'~.
. .
' .

:`~`.. ` :
. ~
~;; - 29 -. .

~ . .

Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In apparatus for analyzing a sample by fluorometric techniques, a carrier member having a surface portion adapted for receiving said sample and including means for supporting said sample thereon as an exposed layer presenting a surface for analysis, test stage means for supporting said member at an analysis location in said apparatus, said carrier member and said stage means being constructed and arranged so that said member is removably insertable into said stage means, illumination means including a source of excitation radiation and means forming a first optical system intersecting said analysis location for delivering said radiation to the surface thereat, optical fluorescence collection means including a photo-detector and means forming a second optical system intersecting said analysis location for receiving the fluorescence emitted from the exposed layer thereat, emission filter means associated with said fluorescence collection means for restricting the sensitivity thereof to a frequency band overlapping the emission spectra of said fluoro-metrically active substance and being substantially non-responsive to the band of excitation radiation, means juxtaposed with said excitation filter means for forming a beam splitter for removing a portion of the light therefrom for developing a reference channel in parallel with the illumination means, means for alternating said illumination means and said reference means: during mutually exclusive periods of time, sensing means for developing a signal indicative of sample illumination on-times when said photodetector is activated by sample fluorescence and for developing a second signal indicative of the period when said reference means. is detected, a demultiplexer response to said signal for alternately delivering outputs indicative of said sample signal or said reference signal, a sample signal processor for receiving said sample signal, a reference signal processor for receiving said reference signal, said processors serving to develop signal outputs, the strength of which is proportional to the amount of light signal received at said photodetector during said sample and reference periods, respectively, a ratio circuit for obtaining the ratio S/R where S equals sample signal and R equals said reference signal, and display means responsive to said ratio circuit for dis-playing the value of said ratio.

2. An apparatus as in claim 1 together with excitation filter means associated with said illumination means for limit-ing the excitation radiation to a predetermined frequency band overlapping the absorption band of said fluorometrically active substance and being substantially non-emissive in the band of fluorometric emission of said substance.

3. An apparatus as in claim 1 together with means disposed in said reference means for forming a diffuse screen therein, and means for collecting light from a portion of said diffuse screen and for inserting the same into said second optical system subsequent to said emission filter means.

4. An apparatus as in claim 1 in which said display means displays the numerical value of said ratio in digital form.

5. An apparatus as in claim 1 in which said alternating means comprises means for alternately blocking and opening said illumination means and said reference means.