CA1281204C

CA1281204C - Fluorometer

Info

Publication number: CA1281204C
Application number: CA000511951A
Authority: CA
Inventors: Walter B. Dandliker; Howard S. Barr; Henry S. Katzenstein; W. Keith R. Watson
Original assignee: Diatron Corp
Current assignee: Diatron Corp
Priority date: 1985-07-01
Filing date: 1986-06-19
Publication date: 1991-03-12
Anticipated expiration: 2008-03-12
Also published as: EP0207485A3; DE3681208D1; US4877965A; EP0207485B1; EP0207485A2; JPS6217643A; JPH0723870B2

Abstract

FLUOROMETER
Abstract of the Invention A fluorometer for measuring a particular fluorescence emanating from a specimen,including producing a burst of concen-trated light energy and directing the concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence. Preferably producing an image of the fluorescence. Detecting the fluorescence and producing a signal in accordance with the fluorescence. Con-trolling the passage of the image of the fluorescence for detecting within a particular time period so as to optimize the detection of the particular fluorescence. Timing the operation to sequence the detection of the fluorescence within the particular time period after the production of the burst of concentrated light energy. Scanning the fluorescence from the specimen for forming signals representative of the fluorescence from the specimen. Analyzing the signals to enhance the portion of the signal representing the particular fluorescence relative to the portion of the signal.

Description

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BACKGROUND OF TllE INVENTION

2 Field of the Invention 4 The present invention relates to fluorometers for detecting a particular fluorescence from a specimen. Specific-6 ally, the present invention relates to a time gated fluorometer 7 for forming an output signal preferably representative of an 8 image of the fluorescent specimen and to a specific method to 9 enhance the analysis of the output signal received to thereby enhance the detection of the particular fluorescence.

12 Description of the Prior Art 14 In general, prior art fluorometers all suffer from a common problem of providing for a discrimination between the 16 generated fluorescent signal and the background noise. Certain 17 types of conventional fluorometers discriminate between the 18 fluorescent signal and the background noise on the basis of wave 19 length. This type of discrimination is 9enerally not sufficient for many types of fluorescent signals.

22 Another type of discrimination can be accomplished 23 using a time gated techniaue. In particular, these instruments 24 are based on the principle of permitting the observation of the fluorescence or luminescence a short, and if desired a variable 26 time, after the excitation period. Time gated fluorometers 27 therefore add an additional level of discrimination b~ viewing 28 the signal fluorescence during an optimal time windowO In the 29 past, this technique generally employed a fluorophore of long 1 decay time in order to allow the background fluorescellce to 2 decay.

4 The time gated techniq~e is in general based on a phosphoroscope invented by Becquerel in 1867. In the Becquerel 6 instrument, the luminescent substance is placed between two 7 rotating discs which are mounted on a common axis and which have 8 sector shaped apertures. The variable time gating is achieved g by an adjustment of the angle between a sector on one disc and a sector on the other. Subsequent refinements of the time gating 11 technique have been accomplished by the use of spark discharges, 12 oscilloscopes, Kerr cells and supersonic cells.

14 The rotating disc invented by Becquerel was put into a conical configuration for a microscope by Jones as described in 16 U.S. Patent No~ 2,071,408 in 1937. Other more recent improve-17 ments have used electronic techniques. For example, l~ieder, 1~ U.S. Patent NO. 4,341,957, provided for the gating of a detect-19 ing circuit electronically and used a laser for excitation. In this way, as in other refinements of Becquerel phosplloroscope, 21 the gating mechanism may be adjusted so that observation of the 22 desired signal can be optimized. Other prior art devices such 23 as Mueller, U.S. Patent No. 4,006,360, use electronic gating to 24 distinguish between species of differing decay time where two species are involved and one is bound dye and the other is an 26 unbound dye.

28 Two commercial instruments are currently available for 29 the measurement of decay time or lifetimes~ Both of these instruments utilize nanosec. flash sources (electric s~ar~ in .~1 ~81204 1 air at reduced pressure). One instr~ment puts the outp~t of a 2 photomultiplier tube onto a fast ocilloscope. Provision is made 3 to match the experimental curve with a sum of up to 3 or 4 4 exponentials.

6 The second instrument excites the sample by repeated 7 flashes from the source (such as at 5kHz) and pulses the photo-8 multiplier at increasingly longer times after the flash. The g output is fed into a recorder or computer and gives an intensity vs. time signal. In addition, this instrument is supplied with 11 software to reconvolute the experimental curve by a well known 12 method termed Linearized Least Squares Reconvolution.

14 Both wavelength based discrimination and time based discrimination suffer by having background fluorescence 16 superimposed on the signal with only an indirect means of 17 segregating the two. In addition, the use of dyes of long decay lB time effectively smears out the desired signal over a long time 19 period, thus making this signal hard to extract. Dyes of long decay time have inherently low extinction coefficients and 21 therefore provide inefficient excitation of the fluorescence~

The present invention is directed to a new type of 26 fluorometer which permits the signal from the fluorophore to be 27 automatically separated from the background in an improved 28 manner to produce an enhanced fluorescent signal. This enhance~
29 ment of the fluorescent signal occurs by a particular instrument 128~2~4 1 desi9n, by the type of data which is collected and l-y a specific 2 method used to process this data.

4 In particular, in a specific embodiment, the invention is implemented using optical time gating wherein the time gating 6 allows high quality optical imaging of the fluorescent source 7 and operates at speeds consistent with the use of common 8 fluorescent labels. This is accomplished using electro-optic 9 modulators which are made of crystals which are cubic and hence isotropic when unstressed. Such modulators operate with large 11 numerical apertures, are fast and therefore are suitahle for use lZ in a gated fluorescence microscope version of the present 13 invention. As an example, the modulators may be made of a cubic 14 crystal material such as Cu Cl (cuprous chloride~. Tl-e use of such electro-optic modulators makes possible the construction of 16 a gated fluorescence microscope in which the optical image is 17 viewed directly such as in normal microscopy.

19 The use of high quality optical imaging is not necessary if the invention is used only as a fluorometer for 21 detecting the fluorescent signal as opposed to providing a 22 representation of the fluorescent image. However, even in the 23 former case imaging is advantageous since such ima9ing provides 24 for an efficient collection of light.

26 In one actual embodiment of the present invelltion, the 27 fluorometer may be considered to be a one pixel display. The 28 present invention pro~ides for an enhanced optical image of a 29 fluorescent specimen formed from a larye number of ~ixels. In one embodiment a single pulse from a laser is used to excite \

~28~L204 1 fluorescence from a microscopic spot on the specimen. The 2 position of the spot on the specimen depends upon the position 3 of a stepping stage all of which is sensed and under the control 4 of control circuitry.

6 A microscope objective focuses the light from the 7 excited spot onto a photodetector through an electro- optic modulator or an equivalent structure such as an electronically g time gated photodetector. The electro-optic modulator is used as an optical shutter. The electro-optic modulator is designed 11 to have a high numerical aperture to thereby allow the collec-12 tion of more light over a greater solid angle thereby permitting 13 a higher resolution in the image formation. The present inven-14 tion also improves the image quality to thereby provide a better signal to noise ratio than other types of electro-optic modula-16 tors such as Pockels cells or Kerr cells. In addition, the 17 collection of more light is benefical to improve the signal to 18 noise ratio relative to a photodetector positioned after the 19 electro-optic modulator. The electro-optic modulator opens at a time t~ after the burst from the laser and closes at a time 21 t~ . The timing control of the opening and closing of the 22 electo- optic modulator and the control of the laser, is 23 provided by the control circuitry.

The fluorescence may be recovered by a photodetector 26 and the recovered information may be stored and processed to 27 extract from the fluroescent signal, intensity as a function of 28 time, and with the information for the one illuminated spot 29 stored for subsequent display. The stepping stage may now move the specimen to a different spot as directed by the control cir-~1 i28~204 1 cuitry and the above process is repeated until the specimen has 2 been scanned as desired to build up a complete enhanced image.
3 If desired, the stored information may be printed out in a 4 numerical form as opposed to providing for an actual image and in such a generalized case, the instrument of the present inven-6 tion is characterized as a fluorometer rather than the 7 specialized designation of fluoromicroscope.

g In another embodiment of the invention~ the image of a large number of pixels is directly transmitted through the 11 electro-optic modulator to a photosensitive array. The array is 12 then scanned to form the complete image of the fluorescence from 13 the specimen.

The advantages present in the apparatus and method of 16 the present invention are based on a number of physical princi-17 ples which underlie the instrument design and method of 18 operation. In particular, these physical principles include the 19 excitation by a concentrated pulse or burst of li~ht energy pro-vided by the laser which is very short in duration compared to 21 the decay time of the fluorophore of interest. In addition, the 22 excitation provided by the laser produces a light pulse of 23 sufficient energy to excite substantially all of the fluoro-24 phore molecules so that substantially all of the flourescence of interest is at its peak excitation when the burst ends. If the 26 burst is too long, then the fluorescense of interest starts to 27 decay during excitation thereby losing signal and contributing 28 indirectly to photobleaching. If the burst is of insufficient 29 energy then random fluctuations in both the fluorescence photon ~8~

1 flux and electrical dark noise become more important and affect 2 measurement o~ the signal adversely.

he present invention also includes sensing of the fluorescence by means of a photosensitive device gated by an 6 electro-optic modulator to respond promptly after the excitation 7 pulse and arranged to sense the total emmission intensity over a 8 large portion of the entire ti~e course of the particular g fluoresence being measured~ The output of the photosensor is then recorded following the excitation pulse and with this 11 recording provided by suitable fast analog or digital means.

13 The emission resulting from the excitation pulse is 14 analyzed by a particular method which permits the particular fluorescent signal, which has a characteristic decay time, to be 16 extracted frcm the time dependence of the total emmission 17 intensity.
1~ .
19 The above described conditions assure that the maximum possible fluorophore signal is obtained while minimizing photo-21 bleaching or fading. In addition, the output signal from the 22 fluorometer or image of the fluoromicroscope of the present 23 invention ideally consists of signals from the fluorophore only.
24 The degree to which this ideal can be approached depends only upon the accuracy with which the curve of intensity versus time 26 for the particular fluorescence can be sensed and recorded.

28 The present invention therefore provides for an 29 apparatus and method of enhancing the sensing of particular fluorescent data and with such enhancement provided both by the 3~8~2~L

1 structural components in the system for detecting the fluores-2 cence, and a method of analysis of the fluorescent signal when 3 detected.

BRIEF DESCRIPTION OF THE DRAWINGS

7 A clearer ~nderstanding of the present invention will 8 be had with reference to the following descriptions and drawings g wherein:

11 Figure 1 illustrates a generalized fluorescent decay 12 curve after excitation of a fluorescent specimen;

14 Figure 2 illustrates a first embodiment of a pulsed 1 ight source;

17 Figure 3 illustrates a second embodiment of a pulsed 18 light source;

Figure 4 illustrates a first embodiment of a 21 fluorometer exemplifying instruments which may be formed as a 22 measurement tool;

24 Figure 5 illustrates a second embodiment of a-fluorometer exemplifying instruments which may be formed as a 26 fluoromicroscope and with a photosensitive array;

28 Figure 6 illustrates a third embodiment of a fluoro-29 meter exemplifying instruments which may be formed as a fluoromicroscope and with a stepping stage; and ~2812~L

1 Figures 7 (a), Ib~ and (c) illustrate various 2 alternative structures for providing direct scanning of the 3 specimen for producing X-Y movement of the beam from the pulsed 4 light source; and 6 Figure 8 is a block diagram of a method of analysis of 7 the fluorescent signal.

9 DESC~IPTION OF THE PREFERRED EMBOD~M~NTS
11 Fluorescent molecules are used as a label or tracer 12 for a number of reasons and in particular have been used in the 13 medical field. In general, the main problem of obtaining the 14 fluorescent signal from the fluorescent molecule is to separate or segregate the desired signal from the unwanted background ~6 fluorescence.

18 In general, two different characteristics may be used 19 to separate the desired signal from the background. In particu-lar, wave length characteristics and decay time characteristics 21 of the emitting fluorescent molecules can be used to implement 22 the separation between signal and background. As shown in 23 Figure 1, once the fluorescent molecules have been illuminated, 24 such as by a laser pulse, the decay of the fluorescence from the assembly of molecules having a number of different decay times 26 provides a time course of intensity versus time of the general 27 character shown in Figure 1.

29 Using the concept of a time window described above, the fluorescence may be observed between ta and t~ where ta g _ ~L28~

1 and t~ are in the general region of the decay time for the 2 particular label being measured. If the fluoresccnt signal is 3 observed only during this particular time window, the signal 4 from the label is enriched since rapidly decaying fluorescence in the background, not associated with the label, will not be 6 sensed and slowly decaying fluorescence, again not associated 7 with the label, will be cut off after the time window. The 8 excited states of all of the molecules begin to decay g immediately but the long decay time or slowly decaying fluroescence is spread out over a longer interval than the short 11 decay time or rapidly decaying fluorescence.

13 The present invention also provides for a method which 14 may be designated as Hybrid Laplace Transform Amplitude Analysis or amplitude analysis for short for extracting a known decay ~6 fluorescent component (TX ) from a noise background containing 17 a multitude of different components (~ ... TN). By this 18 method the decay curve is anal~zed by differentiating the decay 19 curve at N points (where N is the number of fluorescent components irl the background) and by integrating over N
21 intervals along the decay curve. By this particular method a 22 sufficient number of equations is obtained to solve uniquely the 23 desired particular fluorescent signal.

The method may be carried out over the entire time 26 course of the fluorescent decay or over a selected time window 27 as described above in which the signal has been enriched.
28 Depending upon the particular fluorescence being analyzed, one 29 or the other of the time periods may give the more rapid and ~L28~L2~

1 incisive convergence to a final value of N and the un~nown 2 particular fluorescent defined as IX~) 4 It is to be appreciated that the methods of the present invention can be used in conjunction with well known 6 methods of wavelength discrimination.

8 Given a total fluorescence signal IS(t) as a 9 function of time, represented by a sum of unknown signals as well as the desired signal 11IX(O)e kxt, kx= TX-1 12 where 14IS(t) = IX(t) + ~ Ii(t) (1) ~-1 ~6 kxt ~ -kit 17- IX(0) e + ~ Ii(0) e (2) 18 We need to evaluate IX(0). Notice that we know IS(t) for 19 all time. We know the characteristic decay time ( TX =kX-l) of the fluorescent label. We DO NOT know 21 Ii(0)~ ki 22 and wish to evaluate IX(0). Hence we have (2N+1) unknowns.
23 This requires (2N+l) equations for a unique solution. We obtain 24 one equation from equation (2) at t=0 namely IS(0) = IX(0)+ ~ Ii(0) 26 ~-1 27 Bv differentiating equation (2) at N points and by integrating 2~ equation (2) over N intervals we can solve for IX(0).
29 Consider, for example, just one background noise term, say, 31 ~

1 -k1t I1(0)e Applying the above technique, we get -kXt -k1t Is(t) = Ix(O)e + Il~o)e (4) Differentiating equation (4) with respect to t we get:
-kxt -k1t 8 I'S(t) = -kXIx(O)e -klI1(O)e (5) Integrating equation (4) from 0 to infinity we obtain:

11 -kxt -k1t ~
12 ~(o oo)= -IX(O)e I1(0)e (6) _ _ _ _ 13 _ kx k1 Evaluate equation (4) and (5) at t=0 to give:

16 Is(O) = Ix(O) + I1(0) (7) 18 I's(O) = -kXIX(0) - k1I1(0) (8) Substituting limits into equation (6) [x(O) Il (O) ~ (~Cx~ k k 22 We now have three equations; (7), (8) and (9), in three 23 unknowns, Ix()~ Il(0) and k1.

Next, eliminate I1(0) by multiplying equation (7) by kl and 26 adding to equation (8), dropping (O)'s to give equation (12) 28 klIs = k1IX + k1I1 (10) 29 I's = kXIX ~ k1Il ~28iL2()4 ~ s ~ I s = k1IX ~ kXIx (12) 3 Solving equation (123 for k1 gives:
4 kXIX + I s k1 = ~13) Ix ~ Is Substitute this value of k1 Erom equation (13) into equation (9) = + I1 ( - ) kx kXIX ~ I s t14) ll For I1 substitute the value from equation (7) giving:

l3 ~ = x ( x x s ) Rearranging and solving for Ix gives finally a6 2 kX(Is + ~ I s) 17 Ix 18 Is + kx (2 IS - k~ ~ ) (16) The time window technique method may be combined advantageously with the amplitude analysis method described above in treating particular fluorescent data. This is because the underlying assumption in the analysis is that the observed decay curve (intensity versus time) consists of a sum of independent exponential curves one from the label (x) and N curves arising from the N components in the background. Thus, the intensity Is Observed from the entire sample is equation (1) above. The function IS(t) may be measured directly ~ter a single excitation pulse or it may be deduced from several values of ~_ ~
J Is(t) dt which is evaluated by an integrating ~28120~

1 detector after each of several excitation pulses.

3 Since the decay of all components in the fluorcscence 4 is assumed to be exponential, zero time is arbitrary and may be taken at any point along the curve shown in Figure 1. Therefore 6 any zero time assoc~ated with equation (2) results in e~uation 7 (3) as shown above.
8 Equation (2) may be differentiated as follows:

9 -kXt t~ -kit Iis(t) = -kXIX(o) e + `~ -kiIi(0) e (17) L-l 11 Evaluation of Equation (17) at N different points tl to tN

12 gives N independent equations. Integration of equation (2) over N

13 different intervals gives an additional N equations, for example:

15 ~ ~0~1) = = + ~ 8 The 2N=1 equations are sufficient to solve for Ix (0) by eliminating the N different k's and the N different li (O)'s. A numerical solution for Ix () is accomplished 21 by carrying out a series of computations for different N's e.g.
N = 1, N = 2 etc. When the results of the (n+1)th computation are not significantly different from the nth computation, the 24 process is stopped and that value of Ix(O) is taken as the result.

A second method for the extraction of signal may be termed Normalized Background Analysis (NBA). In this method a background measurement is made on an unlabeled or unstained blank sample of material which is otherwise similar to ~ sample ~28~

1 which is staineA or to be stained. In the usual type of 2 background measurement, the blank or unlabeled sample must be 3 identical in both size and fluorescence characteristics to the 4 labeled sample except that no added label is present. In the NBA method the only requirement is that the background be 6 measured on the same type of material but the amount need not be 7 the same. This feature is of particular importance, e.g., in 8 measuring the total amount of label taken up by a sample of g tissue or by a group of cells in the field of a fluorescence microscope. In such a situation, it is not feasible to prepare 11 a blank identical in size to the stained sample and unless the 12 specimen is fixed it is not feasible to measure before and after 13 staining The NBA method may be developed as follows. As 16 before, the background itensity, Ib, is assumed to consist of 17 a sum of intensities with exponential decays:
18 ~ -kit 19 Ib(t) = ~ Ii(0)e (19) t-l 21 The emission from the stained sample is then of the 22 form:
23 ( ) () kxt ~ ~0) kit (20) 24 ~=1 The factor, , provides for the fact that the amount of sample material in the blank and sample measurements may be different. Writing equation (20) in a more compact form 29 IS(t) = ~ Ib(t) + IX(t) (21) Differentiating gives ~1 ~281204 I's(t) = ~ I'b(t~ ~ I'x~) (22) 4 Eliminating ~ from equations (21)and (22) gives:

6 Is~ b I 5Ib = IXI~b - IbI'x (23) Since 9 Ix = Ix(O) e (24) -kXt 11 I'x = -kXIX(O)e = -kXIx (25) 13 Substituting equation (25) into equation (23) and solving for 14 lX gives:

16 I I'b - I~ Ib (26) 17 Ib + Ibkx 19 true for all t.
Figure 2 illustrates a first embodiment of a pulsed 21 light source for use with the various fluormeters formed as 22 embodiments of the present invention. In Figure 2 a laser 1 23 directs light energy to a beam splitter 2. A first portion of 2A the energy from the beam splitter is directed to a photodetector 25 3 and a second portion is directed to a first prism 4. The 26 photo detector 3 produces an output signal when the laser is on 27 and such signal is coupled to control circuitry 5.

29 Control signals are also applied to the control circuitry 5 so that the laser 1 may be controlled "on" and "off"

20~

1 to produce a burst of light energy of a desired short duration 2 as described above. A second prism 6 is positioned to receive 3 light energy from the first prism 4 and to redirect the light 4 energy back to the first prism 4. The distance bet~een the prisms 4 and 6 may be varied to provide a variable optical time 6 delay so that the burst energy is in proper phase with an 7 optical shutter and/or detector which would be part of a 8 complete instrument.

Figure 3 illustrates a second embodiment of a pulsed 11 light source for use with the various flurometers formed as 12 embodiments of the present invention. In Figure 3, a continous 13 wave or wide pulse source 7 of light directs light energy to a 14 modulator 8. The light source is nearly collimated, monochromatic and polarized and is thereby similar to the output 16 from a laser. The modulator 8 forms an optical shutter under 17 the control of control circuitry 9 to produce a short burst of 18 high energy excitin~ light from the modulator 8. The modulator 19 8 may be formed by an electro-optic modulator or an acusto-optic (A0) modulator.
21 Figure 4 is a first embodiment of a fluorometer 22 forming a measurement tool. In figure 4 a specimen 10 to be 23 analysed is positioned on a surface 12. A pulsed light source 24 14, which may be either the light source formed by the embodiment of Figure 2 or the embodiment formed by the 26 embodiment of Figure 3, directs a burst of exciting light to the 27 specimen 10.

29 The light energy from the light source 14 excits fl~orescence in the specimen. The excited fluorescence emits 12~3120~L

1 energy which is directed to an electro-optic modulator ~ so as 2 to produce a time gating of the emitted flo~rescence. The 3 timing control may be provided from a control signal from the 4 control circuitry 5 or 9 of the pulsed light sources shown in Figure 2 and 3. The electro-optic modulator 26 is controlled to 6 open at a time t~ after the burst from the light source and to 7 close at a time t~ , as shown in Figure 1. The emitted 8 fluorescence is therefore directed to a photomultiplier and 9 digitizer 27 to detect and digitize the output emitted fluorescence only between the times taand t~.

12 The output from the photomultiplier and digitizer 27 13 is then coupled to a signal processing and display unit 29 to 14 analyze the information in accordance with the methods described above and to display the results of this analysis. It is to be 16 appreciated that the methods of analysis may be used with a 17 short period time gated fluorescence output but may also be used 18 for analysis over a longer time period. Also, the electo-optic 19 modulator 26 could be eliminated and the emitted fluoresence from the specimen 10 could be directly applied to the 21 photomultiplier and digitizer 27 if the photomultiplier is gated 22 by electronic means.
23 Figure 5 illustrates a first embodiment of a 24 ~luorescence microscope exemplifying instruments which may be formed by incorporating an electro-optic modulator and which may 26 incorporate the methods of analysis described above. As shown 27 in Figure 5, the specimen 10 to be analyzed is positioned on the 28 stationary surface 12. A pulsed light source 14 is controlled 29 to direct a pulse or burst of concentrated light energy toward a dichroic mirror 16~ The mirror 16 directs the light ellergy ~1 3L2~3~L2~

l through an objective lens 18 to the specimen 10 n Li(~ht filters 2 may be added in the excitation and emission beams to thoro~ghly 3 isolate the fluorescence emission and to limit excitation to a 4 single or narrow band of wavelengths.

6 The light energy from the source 14 excites 7 fluorescence in the specimen. The excited fluorescence thereby 8 produces a fluorescent pattern on the specimen 10. The' 9 objective lens 18 forms an image of the fluorescent specimen at a photo sensitive array 20 be~inning at the time ta after the ll pulse froM the source 14. This time t~ is determined by 12 control circuitry forming part of a timin9 control and data 13 processing module 22. The control circuitry actually controls a 14 power supply 24 which in turn controls the operation of the electro- optic modulator 26. The electro-optic modulator is 16 therefore opened at the time ta to allow the image of the 17 fluorescent specimen to be passed to the photo sensitive array 18 20.

At time tB the electro-optic modulator 26 closes so 21 that the photosensitive array has detected at a plurality of 22 elements in the array, information representing the time 23 integral of the intensity of the fluorescence decay from time ta 24 to time t~ after the flash of the light source 14. It is to be appreciated that the time interval between ta to t~ may be a 26 time window having a relatively short duration as described 27 above or may extend out over the entire time course o the 28 fluorescent decay. The particular time interval choscn would be 29 dependent upon the particular type of specimen being observed.
An array control unit 28 scans each element of the array after 1~3i2~)4 1 the electro-optic modulator 26 closes and for each elcment of 2 the array, the array control unit 28 records the time integral 3 of the intensity ~rom t~ to t ~ .

The modu~e 22 includes a data processing portion and 6 this portion stores the data and then analyzes the stored data 7 in accordance with the methods described above to extract the 8 desired particular fluorescent signal rrom the total intensity g stored si~nal. The particular fluorescent signal is then used to produce an output indication such as a signal image on a 11 display 30 and with this signal image representing the desired 12 particular fluorescent signal from the specimen 10.

14 A photosensitive array or multiple detector may be used in two distinctly different modes for fluorescence 16 measurements. In one, all of the elements of the array view the 17 same point but can be used to generate, over the time course of 18 the signal, different mathematical properties of the signal.
19 These properties may be combinations of the intensity, its derivatives or integrals. In another mode each photosensitive 21 element derives the same type of information but for a different 22 point in an image of the fluorescent sample. In this mode, the 23 sample may have to be excited multiple times in order to obtain 24 the required amount of information to solve equation (2).

26 It is to be appreciated that the specific embodiment 27 of a fluoromicroscope illustrated in Figure 5 is illustrative 28 only and that various adaptations and modifications may be made.
29 For example, an image tube may be used in place of the photosensitive array and the image of the fluorescence viewed - 2n -lX8~204 1 directly by an observer or by a camera or with a direct 2 observation by an observer or through a camera without any 3 intervening detectors. Also, the EO modulator can be omitted if 4 the photosensitive array or image tube is gated by electronic means.

7 Figure 6 illustrates a fluoromicroscope exemplifying 8 instruments which may be formed using a stepping stage.
g Portions of the system of Figure 6 similar to those shown in Figure 4 and 5 are given the same reference character.
11 Specifically, in Figure 6 the specimen 10 is mounted on a 12 stepping stage 32. The stepping stage is assummed to be 13 initially at a first position. The light source 14 is 14 controlled to produce a pulse or burst of light energy to excite fluorescence from a single microscopic spot on the specimen 10.
~6 The light source 14 directs the light energy to the specimen 10 17 by reflecting the light energy from the mirror 16 and through 18 the lens 18. The objective lens 18 focuses the fluorescence 19 from the excited spot on the specimen 10 to a photodetector 34.
The actual control of the fluorescence detected by the 21 photodetector 34 is in accordance with the opening and closing 22 of the electro-optic modulator 26.
23 The electro-optic modulator opens at a time t~ after 24 the laser flash and closes at a time t~ . As described above, the time interval may be a short duration time window or may be 26 a period of time sufficiently long to encompass a large fraction 27 of the entire time course for the fluorescence decay. A module 28 36 provides timing control, data storage and processing.
29 Specifically, as in the embodiment of Figure 5, the light source is controlled to produce the pulse of light energy. At a ~L2~312C~

1 predetermined period of time after the pulse, the electro-optic 2 modulator 26 is controlled through the power supply to open and 3 close and thereby act as a shutter. The information detected by 4 the photodetector represents the intensity as a function of time for the one illuminated spot on the specimen. This information 6 is stored by the module 36.

8 Additionally, the module provides for processing of 9 this stored data from the photodetector in accordance with the ]0 methods of analysis described above to separate the desired 11 particular fluorescent signal from the background fluorescence.
12 The information may then be displayed in the display 30 and with 13 the displayed information representing the information for a 14 large number of spots on the specimen. In particular, the stepping stage 32 is controlled to repetitively step to ~6 different spots. This stepping is under the control of the 17 control circuitry in the module 36. After each step, the 18 illumination of a spot is provided by the light source 14 and 19 ~ith a subsequent extraction of the fluorescent si9nal. The process is repeated until the specimen has been scanned in a 21 desired pattern to produce the output display.

23 It is to be appreciated that the specific embodiment 24 of fluoromicroscope illustrated in Figure 6 i6 illustrative only and that various adaptations and modifications may be made. For 26 example, the E0 modulator may be omitted if the photodetector is ~7 gated by electronic means.

29 In Figures 5 and 6, the information at a plurality of spots on the specimen is detected using two different .~1 ~28120~

1 techniques. Figures 7 (a), (b) and (c) illustrate alternative 2 methods of producing this detection of information at a 3 plurality of spots and with these alternate methods incorporated 4 in a structure such as the fluorometer of Figure 4.
~pecifically, as shown in Figure 4 at the position of the dotted 6 block 60, an X-Y positioner may be used to control the exciting 7 light from the pulsed light source to excite the specimen 10 at 8 a plurality of spots for detection.

Figure 7 (a) illustrates a first embodiment of the 11 scanner 60 incorporating a pair of tilting or rotating mirrors 12 62 and 64 such as galvanometer scanning mirrors each producing 13 one axis of movement of the exciting light from the pulsed light 14 source 14 to produce the X-Y scanning of the specimen 10.
16 Figure 7 (b) illustrates a second embodiment of the 17 scanner 60 incorporating an acoustic-optic (A0) modulator 66 and 18 a tilting or rotating mirror 68, each producing one axis of 19 movement of the exciting light from the pulsed light source 14 to produce the X-Y scanning of the specimen 10.

22 Figure 7 (c) illustrates a third embodiment of the 23 scanner 60 incorporating a pair of A0 modulators 70 and 72, each 24 producing one axis of movement of the exciting light from the pulse of light source 14 to produce the X-Y scanning of the 26 specimen 10.
~7 28 In these scanning systems, the scanning is 29 accomplished by movement of the beam in the x and in the y directions so as to systematically illuminate in suecession each l point in a field. If large areas are to be scanned tl~se 2 systems may be combined with the steppin~ stage to provide 3 movement from field to field. Deflections may be accomplished 4 by the galvanometer scanning mirror which reflects the beam in the desired direction or by the acoustic optics (AO~ modulator 6 which deviates the beam into the desired direction.

8 The position of a galvanometer scanning mirror is 9 controlled by the current flow through a coil in a magnetic field. The deviation of a beam by the AO modulator is a 11 function of the frequency applied to the A0 material which then 12 behaves like a diffraction grating. Undeviated light is blanked 13 off optically. In the A0 material, standing waves are set up 14 producing a set of bands of refractive index gradients by which the light is deviated. A sonic transducer in contact with the 16 A0 material produces the periodic mechanical stress within the 17 ~O material.

19 The electro-optic modulator 26 used in the embodiments of Figures 4, 5 and 6 is preferably a modulator of a high 21 numerical aperture to allow the collection and passage of as 22 much light as possible. The use of such an electro-optic 23 modulator provides for an improved signal to noise ratio for the 24 overall system. In general, the electro-optic modulator should have the following characteristics; a high speed which thereby 26 implies a large electro-optic coefficient at gigahertz 27 frequencies together with small power consumption to thereby 28 permit reasonable size power supplies; large angular or 29 numerical aperture which is the most important requirement sinc~
the numerical apeture of the optical system should not be less 2~4L

l than that of a good microscope objective. The large numerical 2 aperture is therefore desirable to a obtain the needed optical 3 resolution to permit formation of a high quality optical image.

It has been generally known that crystals of the cubic 6 class Td (or 43m) offer the maximum angular aperture for 7 devices based on longitudinal or transverse Pockels effects.
8 The following factors are generally involved in the choice of g the particular material to be used in the electro-optic modulator of the present invention. Specifically, when an ll electric field is applied to a cubic crystal (isotropic) of a 12 class Td the crystal becomes birefringent. In general, it 13 becomes biaxial and maximum retardation is obtained for light in 14 the 110 direction and field in the 110 direction. If the field is applied in the 111 direction the crystal becomes uniaxial, 16 the 111 direction being the optic axis. A light beam passing in 17 any direction perpendicular to the 111 direction has a 18 retardation ~ times the maximum retardation mentioned above.
l9 The use of the latter (transverse) mode has the advantage that 2~ the electrodes on the modulator need not be transparent thereby 21 allowing low resistivity to be easily obtainable~ The following 22 group of cubic crystals belong to a group from which the 23 electro- optic modulator of the present invention may be 24 constructed. These cubic crystals include:

26 CuCl (Cuprous chloride) 27 CuBr (Cuprous bromide) 28 CuI (Cuprous iodide) 29 ZnS (Zinc sulfide) ZnSe (Zinc selenide) ~1 2~

1 ZnTe (Zinc telluride) 2 (CH2)6N4 (He~amine or Hexarnethylenetetramine) (Nal Ca)8-4(SO4)2-1[(AlSiO4)6] (Hauynite) 4 GaP (Gallium phosphide) Bi4(Ge04~3 (~ismuth germanate) 6 NaClO3 (Sodium chlorate) 7 BaTiO3 (Barium titanate) 8 SrTiO3 (Strontium titanate) g KTaO3 (Potassium tantalate) KTaxNb1_x03 (Potassium tantalate niobate) 12 The use of electro-optic modulators, formed by cubic 13 crystals of the class Td providing for the time gating, allow 14 for a high quality optical imaging of the fluorescent source from the specimens. These modulators operate with large 16 numerical apertures and are therefore suitable for use in the 17 gated fluorescent microscope of the present invention. The use 18 of these electro-optic modulators makes possible the production 19 of an optica:L image that can be viewed in a similar way to normal microscopy.

22 As disclosed above, these electro-optic modulators may 23 be incorporated in the two embodiments of a fluoromicroscope 24 shown in Figures 5 and 6. In addition, the output fluorescent signal may be enhanced using the unique methods of analysis of 26 the fluorescent data to further separate this data from the 27 background fluorescence. Specifically, as shown in Fiyure 8 one 28 of the rnethods extracts a particular desired fluorescent signal 29 having a known decay from a background of unknown noise signals.
30 The method encompasses differentiating the composite signal at a .~1 , - 2~ -~8~2~)4 1 predetermined number of time points as shown in block 50 and 2 integrating the composite signal over a predetermined number of 3 time intervals as shown in block 52. A computer 54 is then used 4 to eliminate the unknowns using the multiple equations formed by the differentiation and integration to thereby extract the 6 intensity of the desired fluorescent decay signal.

8 The present invention therefore provides for an g apparatus and method of producing an improved detection of fluorescent signals and provides for discrimination between the 11 desired fluorescent signal and the background noise.

13 The present invention provides for excitation by a 14 light pulse very short compared to the decay time of the fluorophore. The light pulse is also of sufficient energy to 16 excite all, or nearly all of the fluorophore molecules in the 17 illuminated sample. The light pulse may be produced by a number 18 of different means. For example, the invention provides for the 19 production of a short, high energy pulse of light by the use of a pulsed laser. In addition, the invention may provide for the 21 short, high energy pulse of light by means of an intense 22 continuous source or wide pulse source in conjunction with an 23 optical shutter. The shutter for example, may be an 24 electro-optic modulator or an AQ modulator.

26 The fluorophore signal contained in the tot~l observed 27 fluorescence may be enhanced, as compared to the background 28 fluorescence, by means of time gating. The time gating may be 29 implemented by a variety of different means. For example, an electronically gated photomultiplier tube or other suitable ~L~8~04 1 photodetector may provide the time gating. Other suitable 2 photodetectors may include an electronically gated image tube OL-3 an electronically gated photosensitive array. The time gating 4 may also be provided by an optical shutter. For example, a Pockels cell may be used to provide an optical shutter. In 6 addition, other electro-optic modulators may be used to provide 7 a shutter and the invention specifically provides for the use of 8 modulators made from cubic crystals of the class Td as optical g shutters having large numerical apertures.

11 The various embodiments of the invention may provide 12 for the detection of the fluorophore signal from a variety of 13 different types of detectors. For example, detectors such as 14 photo-multipliers, image tubes or photosensitive arrays may be used to detect the fluorescence of interest. In addition, 16 microscope optics may be used to orm an image of the 17 fluorescence sample with such microscope optics forming part of 18 the detector.

Once the fluorescence of interest is detected, the 21 signal may be electronically processed in a variery of different 22 ways. In particular, a photosensitive array may be 23 electronically scanned so as to determine for each pixel the 24 fluorescence intensity averaged over the duration of an arbitrary time window. Another processing technique would be 26 measurement of the fluorescence from a sample so as to determine 27 from each pixel the fluorescence intensity averaged over the 28 duration of an arbitrary time window, or as a function of time 29 over a time window. In the processing, the fluorescence intensity may be digitized with any of the processing 31 techniaues. Other aspects of the processing could be the 32 measurement of the fluorescence intensity as a function of time . ~

~23~12~4 1 after a single excitation pulse, or the measurment o~~ the tirne 2 integral of the fluorescence intensity after each of a number oL
3 excitation pulses, and with the integration carried out over a 4 different time interval for each pulse.

6 After the fluorescence of interest has been detected and 7 processed, it may now be analyzed using one of the methods of 8 the present invention. Specifically, the fluorophore signal may 9 be extracted from the total observed fluorescence by means of Hybrid Laplace Transform Amplitude Analysis or by Normalized 11 Background Analysis. In addition, a reconstruction of an image 12 of the fluorescence sample may be produced from the digital data 13 relating to the fluorescence intensity.
1~ .
Although the invention has been described with ~6 reference to particular embodiments, it is to be appreciated 17 that various adaptations and modifications may be made and the 18 invention is only to be limited to the appended claims.

3~

_ ~4 _

Claims

1. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including, means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence and to enhance the detection of the particular fluorescence relative to the total fluorescence, the beginning of the particular time period being defined by a first particular time after the burst of concentrated light energy and during the production of the particular fluorescence and the end of the particular time period being defined by a second particular time after the first particular time where the second particular time occurs during the production of the particular fluorescence from the specimen as a result of the burst of the concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the above means to sequence the detection of the fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, means responsive to the signals produced in accordance with the detection for analyzing the signals as a function of time to produce output signals representative of the particular fluorescence from the specimen, and means responsive to the output signals representing the particular fluorescence from the specimen during the particular time period for producing an image of the particular fluorescence.

2. The fluorometer of claim 1 wherein the means for forming the detected signals includes means for producing a scanning of the particular fluorescence from the specimen during the particular time period at different positions on the specimen.

3. The fluorometer of claim 2 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to obtain a reproduction of the detected fluorescence of the different spots on the specimen.

4. The fluorometer of claim 2 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

5. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the concentrated light energy for directing the concentrated light energy toward the specimen to produce fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence for detecting the fluorescence and for producing signals in accordance with the fluorescence, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence and wherein the controlling means is an electro-optic modulator formed by a cubic crystal of the class Td, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the above means to sequence the detection of the fluorescence within the particular time period after the production of the burst of concentrated light energy, and data processing means responsive to the signals produced by the detecting means for analyzing the signals at progressive time points as a function of time to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence.

6. The fluorometer of claim 5 additionally including means for producing a scanning of the fluorescence from the specimen for forming signals representative of the fluorescence from the specimen and the data processing means includes means for differentiating the signals at progressive time points in the particular time period and for integrating the signals at progressive time points in the particular time period and for processing the differentiated and integrated signals at such progressive time points in a particular relationship to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence.

7. The fluorometer of claim 6 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

8. The fluorometer of claim 6 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

9. The fluorometer of claim 5 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the detecting means within a particular time period by detector responsive means and additionally including means for forming signals including a means for producing a scanning of the fluorescence from the specimen.

10. The fluorometer of claim 9 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

11. The fluorometer of claim 9 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

12. The fluorometer of claim 5 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and additionally including means for forming signals including means for observing or recording of the fluorescence from the specimen.

13. The fluorometer of claim 12 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

14. The fluorometer of claim 12 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

15. The fluorometer of claim 12 wherein the means for detecting includes the camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

16. The fluorometer of claim 5 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the detecting means within a particular time period by detector responsive means and additionally including means for forming signals including means for observing or recording of the fluorescence from the specimen.

17. The fluorometer of claim 16 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

18. The fluorometer of claim 16 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

19. The fluorometer of claim 5 wherein the means for producing the burst of concentrated light energy is a laser.

20. The fluorometer of claim 5 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

21. The fluorometer of claim 20 wherein the cubic crystal of the class Td is cuprous chloride.

22. The fluorometer of claim 20 wherein the electro-optic modulator is formed from at least one of the following group of materials:
CuC1 (Cuprous chloride) CuBr (Cuprous bromide) CuI (Cuprous iodide) ZnS (Zinc sulfide) ZnSe (Zinc selenide) ZnTe (Zinc telluride) (CH2)6N4(Hexamine or Hexamethylenetetramine) (Na, Ca)8-4(SO4)2-1[AlSiO4)6] (Hauynite) GaP(Gallium phosphide) Bi4(GeO4)3(Bismuth germanate) NaClO3(Sodium chlorate) BaTiO3(Barium titanate) SrTiO3(Strontium titanate) KTaO3(Potassium tantalate) KTaxNb1-xO3(Potassium tantalate niobate)

23. The fluorometer of claim 5 wherein the time period is relatively long and has an ending time sufficiently long to have substantially all of the fluorescence decayed to a low value relative to the original value of fluorescence.

24. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the concentrated light energy for directing the concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence for detecting the fluorescence and for producing signals in accordance with the fluorescence, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence and wherein the controlling means is an electro-optic modulator formed by a cubic crystal of the class Td, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the above means to sequence the detection of the fluorescence within the particular time period after the production of the burst of concentrated light energy, and means coupled to the detection means for analyzing the detected fluorescence at progressive time points as a point function of time to form signals representative of the particular fluorescence from the specimen.

25. The fluorometer of claim 24 wherein the cubic crystal of the class Td is cuprous chloride.

26. The fluorometer of claim 24 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and with the means for forming signals including a means for producing a scanning of the fluorescence from the specimen.

27. The fluorometer of claim 26 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

28. The fluorometer of claim 26 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

29. The fluorometer of claim 24 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and the controlling means including detector responsive means for controlling the detecting means within a particular time period and the means for forming signals including means for producing a scanning of the fluorescence from the specimen.

30. The fluorometer of claim 29 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

31. The fluorometer of claim 29 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

32. The fluorometer of claim 24 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and with the means for forming signals including means for observing or recording of the fluorescence from the specimen.

33. The fluorometer of claim 32 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

34. The fluorometer of claim 32 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

35. The fluorometer of claim 32 wherein the means for detecting includes a camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

36. The fluorometer of claim 24 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the detecting means within a particular time period by detector responsive means and with the means for forming signals including means for observing or recording the fluorescence from the specimen and the means for forming signals including means for differentiating the detected fluorescence at progressive times during the particular time period and for integrating the detected fluorescence at progressive times during the particular time period and for processing the differentiated fluorescence and the integrated fluorescence at such progressive times during the particular time period to form the signals representative of the particular fluorescence from the specimen.

37. The fluorometer of claim 36 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

38. The fluorometer of claim 36 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

39. The fluorometer of claim 24 wherein the means for producing the burst of concentrated light energy is a laser.

40. The fluorometer of claim 24 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

41. The fluorometer of claim 24 wherein the time period has a beginning time and an ending time defining a sufficiently short time to enhance the detection of the particular fluorescence relative to the total fluorescence.

42. The fluorometer of claim 24 additionally including data processing means responsive to the signals produced by the detecting means for analyzing the signals to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence.

43. The fluorometer of claim 42 wherein the time period is relatively long and has an ending time sufficiently long to have substantially all of the fluorescence decayed to a low value relative to the original value of fluorescence.

44. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore moles in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the concentrated light energy for directing the concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence for detecting the fluorescence and for analyzing the detected fluorescence as a function of time to produce signals in accordance with the fluorescence, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence and wherein the controlling means is an electro-optic modulator formed from at least one of the following group of materials:
CuCl (Cuprous chloride) CuBr (Cuprous bromide) CuI (Cuprous iodide) ZnS (Zinc sulfide) ZnSe (Zinc selenide) ZnTe (Zinc telluride) (CH2)6N4(Hexamine or hexamethylenetetramine) (Na, Ca)8-4(SO4)2-1[(AlSiO4)6] (Hauynite) GaP (Gallium phosphide) Bi4(GeO4)3 (Bismuth germanate) NaC1O3 (Sodium chlorate) BaTiO3 (Barium titanate) SrTiO3 (Strontium titanate) KTaO3 (Potassium tantalate) KTaxNb1-xO3 (Potassium tantalate niobate)

45. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the concentrated light energy for directing the concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence for detecting the fluorescence and for producing signals in accordance with the fluorescence, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the above means to sequence the detection of the fluorescence within the particular time period after the production of the burst of concentrated light energy, and means coupled to the detection means for forming signals representative of the particular fluorescence from the specimen, and data processing means responsive to the signals produced by the detecting means for analyzing the signals to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence, and wherein the data processing means includes first means for differentiating the signals produced by the detecting means at a number of time points to produce a plurality of individual time point signals, second means for integrating the signals produced by the detecting means over a corresponding number of time intervals to produce a plurality of individual time integrated signals and third means responsive to the signals from the detecting means, the individual time point signals and the individual time integrated signals for operating upon these signals in a particular relationship to determine the particular fluorescence from the specimen during the particular period as a result of the burst of concentrated light energy.

46. The fluorometer of claim 45 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and with the means for forming signals including a means for producing a scanning of the fluorescence from the specimen.

47. The fluorometer of claim 4 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

48. The fluorometer of claim 46 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different position.

49. The fluorometer of claim 45 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and the controlling means including detector responsive means for controlling the detecting means within a particular time period and the means for forming signals including means for producing a scanning of the fluorescence from the specimen.

50. The fluorometer of claim 49 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

51. The fluorometer of claim 49 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

52. The fluorometer of claim 45 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and with the means for forming signals including means for observing or recording of the fluorescence from the specimen.

53. The fluorometer of claim 52 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

54. The fluorometer of claim 52 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

55. The fluorometer of claim 52 wherein the means for detecting includes a camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

56. The fluorometer of claim 45 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the detecting means within a particular time period by detector responsive means and with the means for forming signals including means for observing or recording of the fluorescence from the specimen.

57. The fluorometer of claim 56 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

58. The fluorometer of claim 56 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

59. The fluorometer of claim 45 wherein the means for producing the burst of concentrated light energy is a laser.

60. The fluorometer of claim 45 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

61. The fluorometer of claim 45 wherein the means for controlling the detecting means within a particular time period is an electro-optic modulator.

62. The fluorometer of claim 45 wherein the time period has a beginning time and an ending time defining a sufficiently short time to enhance the detection of the particular fluorescence relative to the total fluorescence.

63. The fluorometer of claim 45 wherein the time period is relatively long and has an ending time sufficiently long to have substantially all of the fluorescence decayed to a low value relative to the original value of fluorescence.

64. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including, means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means during a particular time period to optimize the detection of the particular fluorescence and wherein the time period has a beginning time and an ending time to enhance the detection of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence, the beginning time of the time period being a first particular time after the burst of concentrated light energy and after the production of the particular fluorescence, and the ending time being a second particular time after the first particular time and during the production of the particular fluorescence from the specimen as a result of the burst of concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, and data processing means responsive to the signals produced by the detecting means during the particular time period for analyzing the signals as a function of time on a point-by-point time basis to enhance the portion of the signals representing the particular fluorescence during the particular time interval relative to the portion of the signals representing the remaining fluorescence during the particular time interval.

65. A fluorometer as set forth in claim 64 wherein the detecting means includes means for scanning the fluorescence from the specimen to obtain the production of the signals representative of the fluorescence from the specimen as a result of the production of the burst of concentrated light energy and wherein the data processing means differentiates the signals as a function of time on a point-by-point time basis and integrates the signals as a function of time on a point-by-point time basis and combines the differentiated and integrated signals in a particular relationship to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence during the particular time interval.

66. The fluorometer of claim 65 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

67. The fluorometer of claim 65 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

68. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the concentrated light energy for directing the concentrated light energy toward the specimen to produce fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence for detecting the fluorescence and for producing signals in accordance with the fluorescence, means coupled to the detecting means for controlling the detecting means within a particular time period to optimize the detection of the particular fluorescence, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the above means to sequence the detection of the fluorescence within the particular time period after the production of the burst of concentrated light energy, data processing means responsive to the signals produced by the detecting means for analyzing the signals to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence, and the data processing means including first means for differentiating the signals produced by the detecting means at a number of time points to produce a plurality of individual time point signals, second means for integrating the signals produced by the detecting means over a corresponding number of time intervals to produce a plurality of individual time integrated signals and third means responsive to the signals from the detecting means, the individual time point signals and the individual time integrated signals for producing signals representing the particular fluorescence.

69. The fluorometer of claim 68 additionally including means for producing a scanning of the fluorescence from the specimen for forming signals representative of the fluorescence from the specimen.

70. The fluorometer of claim 69 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

71. The fluorometer of claim 69 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

72. The fluorometer of claim 69 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and the controlling means including detector responsive means for controlling the detecting means within a particular time period.

73. The fluorometer of claim 72 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

74. The fluorometer of claim 72 wherein the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

75. The fluorometer of claim 68 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the passage of the image of the fluorescence to the detecting means and additionally including means for forming signals including means for observing or recording of the fluorescence from the specimen.

76. The fluorometer of claim 75 wherein the means for detecting includes a photodetector for detecting the fluorescence from the specimen.

77. The fluorometer of claim 75 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

78. The fluorometer of claim 75 wherein the means for detecting includes the human eye or a camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

79. The fluorometer of claim 68 additionally including means responsive to the fluorescence from the specimen for producing an image of the fluorescence and with the controlling means controlling the detecting means within a particular time period by detector responsive means and additionally including means for forming signals including means for observing or recording of the fluorescence from the specimen.

80. The fluorometer of claim 79 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

81. The fluorometer of claim 79 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

82. The fluorometer of claim 68 wherein the means for producing the burst of concentrated light energy is a laser.

83. The fluorometer of claim 68 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

84. The fluorometer of claim 68 wherein the means for controlling the passage of the image is an electro-optic modulator.

85. The fluorometer of clam 84 wherein the electro-optic modulator is formed by a cubic crystal of the class Td.

86. The fluorometer of claim 85 wherein the cubic crystal of the class Td is cuprous chloride.

87. The fluorometer of claim 85 wherein the electro-optic modulator is formed from at least one of the following group of materials:
CuCl (Cuprous chloride) CuBr (Cuprous bromide) CuI (Cuprous iodide) ZnS (Zinc sulfide) ZnSe (Zinc selenide) ZnTe (Zinc telluride) (CH2)6N4(Hexamine or Hexamethylenetetramine) (Na, Ca)8-4(SO4)2-1[(AlSiO4)6](Hauynite) GaP (Gallium phosphide) Bi4(GeO4)3 (Bismuth germanate) NaClO3 (Sodium chlorate) BaTiO3 (Barium titanate) SrTiO3 Strontium titanate) KTaO3 (Potassium tantalate) KTaxNb1-xO3 (Potassium tantalate niobate)

88. The fluorometer of claim 68 wherein the time period is relatively long and has an ending time sufficiently long to have substantially all of the fluorescence decayed to a low value relative to the original value of fluorescence.

89. The fluorometer of claim 68 wherein the time period has a beginning time and an ending time defining a sufficiently short time to enhance the detection of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence.

90. The fluorometer of claim 68 wherein the first means produces the plurality of individual time point signals produces the plurality of individual time point signals for a specimen not carrying a fluorescent label, said specimen being not necessarily of the same size and disposition as that of the specimen carrying the fluorescent label but consisting of the same material but without added fluorescent label, and the third means extracts from the individual time point signals and the derivatives at the corresponding time points and the characteristic decay time of the particular fluorescence the emission intensity representing the particular fluorescence only and being separated from the background emission.

91. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means in a particular time period to optimize the detection of the particular fluorescence wherein the time period has a beginning time and an ending time to enhance the detection of the particular fluorescence relative to the total fluorescence, the beginning time occurring a first particular time after the burst of concentrated light energy and during the production of the particular fluorescence and the ending time occurring a second particular time after the first particular time and during the production of the particular fluorescence from the specimen as a result of the burst of concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, means coupled to the detecting means for analyzing the detected fluorescence on a differential and integral basis as a function of time to form signals representative of the particular fluorescence from the specimen, and means responsive to the detection of the particular fluorescence from the specimen during the particular time period for producing an image of the particular fluorescence during the particular time interval.

92. The fluorometer of claim 71 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and the means for producing signals includes an array control for scanning the photosensitive array to obtain a reproduction of the detected fluorescence of the different spots on the specimen.

93. The fluorometer of claim 91 including, a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

94. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means during a particular time period to optimize the detection of the particular fluorescence wherein the particular time period has a beginning time and an ending time to enhance the detection of the particular fluorescence relative to the total fluorescence, the beginning time and the ending time occurring during the production of the particular fluorescence from the specimen as a result of the burst of concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the particular fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, means coupled to the detecting means for analyzing the detected fluorescence on a differential and integral basis as a function of time to form signals representative of the particular fluorescence from the specimen during the particular time period, and means responsive to the fluorescence from the specimen for producing an image of the particular fluorescence during the particular time period.

95. The fluorometer of claim 94 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

96. The fluorometer of claim 94 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

97. The fluorometer of claim 94 wherein the means for detecting includes the camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

98. The fluorometer of claim 94 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

99. The fluorometer of claim 94 wherein the means for controlling the detecting means within a particular time period is an electro-optic modulator.

100. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means during a particular time period to optimize the detection of the particular fluorescence during the particular time period, the particular time period having a beginning time and an ending time to enhance the detection of the particular fluorescence relative to the fluorescence including the particular fluorescence, the beginning time and ending time occurring during the production of the particular fluorescence from the specimen as a result of the production of the burst of concentrated energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, means coupled to the detecting means for analyzing the detected fluorescence at progressive instants of time on a differential and integral basis to form signals representative of the particular fluorescence from the specimen, and means responsive to the signals from the detecting means during the particular time period for producing an image of the particular fluorescence during the particular time period.

101. The fluorometer of claim 100 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

102. The fluorometer of claim 100 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

103. The fluorometer of claim 100 wherein the means for producing the burst of concentrated light energy is a laser.

104. The fluorometer of claim 100 additionally including data processing means responsive to the signals produced by the detecting means for analyzing the signals to enhance the portion of the signals representing the particular fluorescence relative to the portion of the signals representing the remaining fluorescence.

105. A fluorometer as set forth in claim 100, including, means for recording the particular fluorescence from the specimen during the particular time period as a result of the burst of concentrated light energy.

106. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce a fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting the fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means during a particular time period to optimize the detection of the particular fluorescence during the particular time period the particular time period having a beginning time and an ending time to enhance the detection of the signals representing the particular fluorescence during the particular time period relative to the portion of the signals representing the remaining fluorescence during the particular time period, the beginning time and the ending time occurring during the production of the particular fluorescence from the specimen as a result of the burst of concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence from the specimen during the particular time period as a result of the production of the burst of concentrated light energy, data processing means responsive to the signals produced by the detecting means for analyzing the signals as a function of time to enhance the portion of the signals representing the particular fluorescence during the particular time interval relative to the portion of the signals representing the fluorescence during the particular time interval other than the particular fluorescence, and means responsive to the signal from the data processing means for producing an image of the particular fluorescence.

107. The fluorometer of claim 106 wherein the means for detecting includes a photosensitive array for detecting the fluorescence emanating from a plurality of spots on the specimen and means are included for scanning the fluorescence from the specimen and the means for scanning includes an array control for scanning the photosensitive array to reproduce the detected fluorescence of the different spots on the specimen.

108. The fluorometer of claim 106 wherein means are included for scanning the fluorescence from the specimen and the means for scanning includes a stepping stage for supporting the specimen and for moving the specimen to a plurality of different positions for providing a detection of the fluorescence emanating from the specimen at the different positions.

109. A fluorometer as set forth in claim 106 wherein the means for forming signals includes means for scanning the fluorescence produced from the specimen during the particular time period as a result of the burst of concentrated light energy.

110. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting such fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means within a particular time period to optimize the detection of the particular fluorescence wherein the particular time period has a beginning time and an ending time to enhance the detection of the signals representing the particular fluorescence during the particular time period relative to the portion of the signals representing the fluorescence during the particular time period other than the particular fluorescence, the beginning and ending times occurring during the production of the particular fluorescence from the specimen as a result of the burst of concentrated energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the particular fluorescence from the specimen during the particular time period as a result of the production of the burst of concentrated light energy, data processing means responsive to the signals produced by the detecting means as an instantaneous function of time during the particular time period for analyzing the signals to enhance the portion of the signals representing the particular fluorescence during the particular time period relative to the portion of the signals representing the fluorescence during the particular time period other than particular fluorescence, and means responsive to the signals from the data processing means for producing an image of the fluorescence.

111. The fluorometer of claim 110 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

112. The fluorometer of claim 106 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

113. The fluorometer of claim 110 wherein the means for detecting includes a camera to analyze or record the fluorescence emanating from a plurality of spots on the specimen.

114. The fluorometer of claim 110 wherein the means for producing the burst of concentrated light energy is a continuous wave source directing light energy through a modulator.

115. A fluorometer as set forth in claim 110, including, means for recording the particular fluorescence produced from the specimen during the particular time period as a result of the burst of concentrated light energy.

116. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce fluorescence from the specimen .

including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting such fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means within a particular time period to optimize the detection of the particular fluorescence wherein the time period has a beginning time and an ending time to enhance the detection of the signals representing the particular fluorescence during the particular time interval relative to the portion of the signals representing the fluorescence during the particular time period other than the particular fluorescence, the beginning and ending times occurring during the production of the particular fluorescence from the specimen as a result of the burst of concentrated light energy, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence within the particular time period as a result of the production of the burst of concentrated light energy, data processing means responsive to the signals produced by the detecting means during the particular time period for analyzing the signals at progressive instants of time during the particular time period on a differential and integral basis to enhance the portion of the signals representing the particular fluorescence during the particular time period relative to the portion of the signals representing the fluorescence during the particular time period other than the particular fluorescence, and means responsive to the enhanced signals from the data processing means for producing an image of the particular fluorescence during the particular time period.

117. The fluorometer of claim 116 wherein the means for detecting includes a photodetector for detecting the fluorescence emanating from the specimen.

118. The fluorometer of claim 116 wherein the means for detecting includes an image tube to reproduce the pattern of fluorescence emanating from a plurality of spots on the specimen.

119. The fluorometer of claim 116 wherein the means for producing the burst of concentrated light energy is laser.

120. A fluorometer as set forth in claim 116, including, means for recording the particular fluorescence produced from the specimen during the particular time period as a result of the burst of concentrated light energy.

121. A fluorometer for measuring a particular fluorescence emanating from particular fluorophore molecules in a specimen, including means for producing a burst of concentrated light energy having a pulse time short compared to the decay time of the particular fluorescence and having sufficient energy to excite substantially all of the particular fluorophore molecules, means responsive to the burst of concentrated light energy for directing the burst of concentrated light energy toward the specimen to produce fluorescence from the specimen including the particular fluorescence, means responsive to the fluorescence from the specimen for detecting such fluorescence and for producing signals in accordance with such detection, means coupled to the detecting means for obtaining a controlled operation of the detecting means within a particular time period to optimize the detection of the particular fluorescence wherein the particular time period has a beginning time and an ending time to enhance the detection of the signals representing the particular fluorescence during the particular time relative to the portion of the signals representing the fluorescence during the particular time period other than the particular fluorescence the beginning and ending times occurring during the production of the particular fluorescence, means coupled to the burst producing means, to the detecting means and to the controlling means for timing the operation of the detecting means to sequence the detection of the fluorescence during the particular time period as a result of the production of the burst of concentrated light energy, data processing means responsive to the signals produced by the detecting means during the particular time period for analyzing the signals at different time points on a differential basis and an integral basis during the particular time period and for combining the results of the differential and integral analyses in a particular relationship to enhance the portion of the signals representing the particular fluorescence during the particular time period relative to the portion of the signals representing the fluorescence during the particular time period other than the particular fluorescence, and the means for controlling the passage of the image constituting an electro-optic modulator formed by a cubic crystal of the class Td.

122. The fluorometer of claim 121 wherein the cubic crystal of the class Td is cuprous chloride.

123. The fluorometer of claim 121 wherein the electro-optic modulator is formed from at least one of the following group of materials:
CuCl (Cuprous chloride) CuBr (Cuprous bromide) CuI (Cuprous iodide) ZnS (Zinc sulfide) ZnSe (Zinc selenide) ZnTe (Zinc telluride) (CH2)6N4(Hexamine or Hexamethylenetetramine) (Na, Ca)8-4(SO4)2-1[(AlSiO4)6](Hauynite) GaP (Gallium phosphide) Bi4(GeO4)3 (Bismuth germanate) NaClO3 (Sodium chlorate) BaTiO3 (Barium titanate) SrTiO3 (Strontium titanate) KTaO3 (Potassium tantalate) KTaxNb1-xO3 (Potassium tantalate niobate)