U.S. patent number RE34,782 [Application Number 07/969,794] was granted by the patent office on 1994-11-08 for fluorometer.
This patent grant is currently assigned to Diatron Corporation. Invention is credited to Howard S. Barr, Walter B. Dandliker, Henry S. Katzenstein, Keith R. Watson.
United States Patent |
RE34,782 |
Dandliker , et al. |
November 8, 1994 |
Fluorometer
Abstract
A fluorometer for measuring a particular fluorescence emanating
from a specimen and operating in accordance with the following
method. Producing a burst of concentrated 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. Controlling 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.
Inventors: |
Dandliker; Walter B.
(Escondido, CA), Barr; Howard S. (Escondido, CA),
Katzenstein; Henry S. (Pacific Palisades, CA), Watson; Keith
R. (Baja California, MX) |
Assignee: |
Diatron Corporation (San Diego,
CA)
|
Family
ID: |
27086634 |
Appl.
No.: |
07/969,794 |
Filed: |
October 12, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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611929 |
Nov 13, 1990 |
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Reissue of: |
751746 |
Jul 1, 1985 |
04877965 |
Oct 31, 1989 |
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Current U.S.
Class: |
250/458.1;
250/461.2; 356/318 |
Current CPC
Class: |
G01N
21/6408 (20130101); G02B 21/16 (20130101); G01N
21/6456 (20130101); G01N 21/6458 (20130101); G01N
2201/1087 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G02B 21/16 (20060101); G01N
021/64 () |
Field of
Search: |
;250/458.1,461.2
;356/318 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2532756 |
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Mar 1984 |
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FR |
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3502059 |
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Nov 1985 |
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DE |
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Primary Examiner: Hannaher; Constantine
Attorney, Agent or Firm: Roston; Ellsworth R. Schwartz;
Charles H.
Parent Case Text
.Iadd.This is a continuation of reissue application Ser. No.
07/611,929 filed Nov. 13, 1990 now abandoned. .Iaddend.
Claims
We claim:
1. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence.Iaddend.,
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 .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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.Iadd., after the burst of the
concentrated light energy, .Iaddend.to optimize the detection of
the particular fluorescence and to enhance the detection of the
particular fluorescence relative to the .[.total.]. .Iadd.detection
of the background .Iaddend.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 .Iadd.and the background
fluorescence .Iaddend.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.]. .Iadd.such .Iaddend.means to sequence the detection of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.during the
particular time period .[.as a result of.]. .Iadd.after
.Iaddend.the production of the burst of concentrated light
energy,
means .[.coupled to the detection means for forming.].
.Iadd.responsive to the signals produced in accordance with the
detection during the particular time period for distinguishing the
particular fluorescence produced during the particular time period
from the background fluorescence produced during the particular
time period and for producing output .Iaddend.signals
representative .Iadd.substantially only .Iaddend.of the particular
fluorescence from the specimen .Iadd.during the particular time
period.Iaddend., and
means responsive to the .[.detection of.]. .Iadd.output signals
representing substantially only .Iaddend.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
signals.]. .Iadd.detecting the fluorescences .Iaddend.includes
means for producing a scanning of the particular .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence, the particular
fluorescence having different characteristics than the background
fluorescence.Iaddend., 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.]. .Iadd.fluorescences .Iaddend.from the specimen
including the particular fluorescence .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence from the specimen
.Iaddend.for detecting .[.the fluorescence .]. .Iadd.such
fluorescences .Iaddend.and for producing signals in accordance with
.[.the fluorescence.]. .Iadd.such detection.Iaddend.,
means coupled to the detecting means for controlling the detecting
means within a particular time period .[.to optimize the detection
of the particular fluorescence.]. .Iadd.after the burst of the
concentrated light energy .Iaddend.and wherein the controlling
means is an electro-optic modulator formed by a cubic crystal of
the class T.sub.d, .Iadd.the fluorescence produced during the
particular time period including the particular fluorescence and
the background fluorescence.Iaddend.,
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 .[.analysing the signals to enhance.].
.Iadd.distinguishing .Iaddend.the portion of the signals
.[.representing.]. .Iadd.produced during the particular time period
in representation of .Iaddend.the particular fluorescence
.[.relative to.]. .Iadd.from .Iaddend.the portion of the signals
.[.representing the remaining.]. .Iadd.produced during the
particular time period in representation of the background
.Iaddend.fluorescence .Iadd.and for processing substantially only
the portion of the signals representing the particular fluorescence
to measure the particular fluorescence.Iaddend..
6. The fluorometer of claim 5 additionally including means for
producing a scanning of the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for forming signals representative of
the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from the
specimen.
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 .Iadd.substantially only .Iaddend.to the .Iadd.signals
representing the particular .Iaddend.fluorescence from the specimen
for producing an image of .[.the.]. .Iadd.such
.Iaddend.fluorescence .[.and with the controlling means controlling
the detecting means within a particular time period by detector
responsive means.]. and .Iadd.the detecting means
.Iaddend.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.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.emanating from a
plurality of spots on the specimen.
15. The fluorometer of claim 12 wherein the means for detecting
includes .[.the.]. .Iadd.a .Iaddend.camera to analyze or record the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.emanating from a
plurality of spots on the specimen.
16. The fluorometer of claim 5 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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 .Iadd.substantially only .Iaddend.the
.Iadd.particular .Iaddend.fluorescence from the specimen.
17. The fluorometer of claim 16 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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 T.sub.d 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:
CuCl (Cuprous chloride)
CuBr (Cuprous bromide)
CuI (Cuprous iodide)
ZnS (Zinc sulfide)
ZnSe (Zinc selenide)
ZnTe (Zinc telluride)
(CH.sub.2).sub.6 N.sub.4 (Hexamine or Hexamethylenetetramine)
(Na, Ca).sub.8-4 (SO.sub.4).sub.2-1 [(AlSiO.sub.4).sub.6 ]
(Hauynite)
GaP (Gallium phosphide)
Bi.sub.4 (GeO.sub.4).sub.3 (Bismuth germanate)
NaClO.sub.3 (Sodium chlorate)
BaTiO.sub.3 (Barium titanate)
SrTiO.sub.3 (Strontium titanate)
KTaO.sub.3 (Potassium tantalate)
KTa.sub.x Nb.sub.1-x O.sub.3 (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.]. .Iadd.fluorescences
.Iaddend.decayed .Iadd.at the end of such time period .Iaddend.to a
low value relative to the original value of .[.fluorescence.].
.Iadd.such fluorescences.Iaddend..
24. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence.Iaddend., 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 .Iadd.and the background fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.for detecting .[.the
fluorescence.]. .Iadd.such fluorescences .Iaddend.and for producing
signals in accordance with .[.the fluorescence.]. .Iadd.such
detection.Iaddend.,
means coupled to the detecting means for controlling the detecting
means within a particular time period .Iadd.after the burst of the
concentrated light energy .Iaddend.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
T.sub.d, the particular fluorescence and the background
fluorescence occurring during the particular time period,
.Iadd.the particular fluorescence and the background fluorescence
having individual decays and the decay of the particular
fluorescence being different from the decay of the background
fluorescence, .Iaddend.
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.]. .Iadd.detecting .Iaddend.means
for .[.forming signals representative of the particular
fluorescence from the specimen.]. .Iadd.distinguishing the decay of
the particular fluorescence during the particular time period from
the decay of the background fluorescence during the particular time
period and for thereby determining the characteristics
substantially only of the particular fluorescence in the
specimen.Iaddend..
25. The fluorometer of claim 24 wherein the cubic crystal of the
class T.sub.d 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.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
29. The fluorometer of claim 24 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences .Iaddend.and the
means for .[.forming.]. .Iadd.producing .Iaddend.signals including
means for producing a scanning of the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen.
30. The fluorometer of claim 29 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
32. The fluorometer of claim 24 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.and with the
controlling means controlling the passage of the image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.to the detecting
means and with the means for forming signals including means for
observing or recording .[.of.]. the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen.
33. The fluorometer of claim 32 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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.].
.Iadd.fluorescences .Iaddend.emanating from a plurality of spots on
the specimen.
36. The fluorometer of claim 24 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences .Iaddend.and with the means
for forming signals including means for observing or recording of
the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from the
specimen.
37. The fluorometer of claim 36 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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 .Iadd.particular
.Iaddend.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.].
.Iadd.background .Iaddend.fluorescence.
42. The fluorometer of claim 24 additionally including data
processing means responsive to the signals produced by the
detecting means for analysing the signals to enhance the portion of
the signals representing the particular fluorescence relative to
the portion of the signals representing the .[.remaining.].
.Iadd.background .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.decayed to a low value relative to the original value of
.[.fluorescence.]. .Iadd.fluorescences.Iaddend..
44. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences .Iaddend.from the specimen
including the particular fluorescence .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.and for
.[.producing.]. .Iadd.analyzing the detected fluorescences as a
function of time to produce .Iaddend.signals .[.in accordance with
the fluorescence.]. .Iadd.distinguishing the particular
fluorescence from the background fluorescence and representing
substantially only the particular fluorescence.Iaddend.,
means coupled to the detecting means for controlling the detecting
means within a particular time period.Iadd., after the burst of the
concentrated light energy, .Iaddend.to optimize the .[.detection
of.]. .Iadd.analysis to distinguish .Iaddend.the particular
fluorescence .Iadd.from the background fluorescence .Iaddend.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 iodine)
ZnS (Zinc sulfide)
ZnSe (Zinc selenide)
ZnTe (Zinc telluride)
(CH.sub.2).sub.6 N.sub.4 (Hexamine or hexamethylenetetramine)
(Na, Ca).sub.8-4 (SO.sub.4).sub.2-1 [(AlSiO.sub.4).sub.6
](Hauynite)
GaP (Gallium phosphide)
Bi.sub.4 (GeO.sub.4).sub.3 (Bismuth germanate)
NaClO.sub.3 (Sodium chlorate)
BaTiO.sub.3 (Barium titanate)
SrTiO.sub.3 (Strontium titanate)
KTaO.sub.3 (Potassium tantalate)
KTa.sub.x Nb.sub.1-x O.sub.3 (Potassium tantalate niobate)
45. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences .Iaddend.from the specimen
including the particular fluorescence .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence from the specimen
.Iaddend.for detecting .[.the fluorescence.]. .Iadd.such
fluorescences .Iaddend.and for producing signals in accordance with
.[.the fluorescence.]. .Iadd.such detection.Iaddend.,
means coupled to the detecting means for controlling the detecting
means within a particular time period.Iadd., after the burst of the
concentrated light energy, .Iaddend.to optimize the detection of
the particular fluorescence .Iadd.relative to the background
fluorescence.Iaddend.,
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 .Iadd.produced
.Iaddend.fluorescence within the particular time period after the
production of the burst of concentrated light energy, and
means coupled to the .[.detection.]. .Iadd.detecting .Iaddend.means
for forming signals representative of the particular fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen,
and
data processing means responsive to the signals produced by the
.[.detecting.]. .Iadd.signal-forming .Iaddend.means .Iadd.during
the particular time period .Iaddend.for .[.analysing the signals to
enhance.]. .Iadd.enhancing .Iaddend.the portion of the signals
representing the particular fluorescence relative to the portion of
the signals representing the .[.remaining.]. .Iadd.background
.Iaddend.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 .Iadd.during the
particular time period .Iaddend.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 .Iadd.during the particular time period
.Iaddend.to produce a plurality of individual time integrated
signals and third means responsive to the signals from the
.[.detecting.]. .Iadd.signal-forming .Iaddend.means, the individual
time point signals and the individual time integrated signals
.Iadd.during the particular time period .Iaddend.for operating upon
these signals in a particular relationship to .Iadd.distinguish the
particular fluorescence from the background fluorescence and to
.Iaddend.determine .Iadd.substantially only .Iaddend.the particular
fluorescence from the specimen during the particular .Iadd.time
.Iaddend.period as a result of the burst of concentrated light
energy.
46. The fluorometer of claim 45 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.and with the
controlling means controlling the passage of the image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.to the detecting
means and with the means for forming signals including a means for
producing a scanning of the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen.
47. The fluorometer of claim 46 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different position.
49. The fluorometer of claim 45 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.such fluorescences .Iaddend.and the
means for forming signals including means for producing a scanning
of the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from the
specimen.
50. The fluorometer of claim 49 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
52. The fluorometer of claim 45 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.and with the
controlling means controlling the passage of the image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.to the detecting
means and with the means for forming signals including means for
observing or recording of the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen.
53. The fluorometer of claim 52 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences .Iaddend.and with the means
for forming signals including means for observing or recording
.[.of.]. the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from
the specimen.
57. The fluorometer of claim 56 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.emanating from the specimen.
58. The fluorometer to claim 56 wherein the means for detecting
includes an image tube to reproduce the pattern of
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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 .Iadd.particular
.Iaddend.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 .].
.Iadd.detection of the background .Iaddend.fluorescence.
63. The fluorometer of claim 45 wherein the .Iadd.particular
.Iaddend.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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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 .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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
.Iadd.after the burst of the concentrated light energy .Iaddend.to
optimize the detection of the particular fluorescence and wherein
the .Iadd.particular .Iaddend.time period has a beginning time and
an ending time to enhance the detection of the .Iadd.portion of the
.Iaddend.signals representing the particular fluorescence relative
to the portion of the signals representing the .[.remaining.].
.Iadd.background .Iaddend.fluorescence, the beginning time of the
particular time period being a first particular time after the
burst of .Iadd.the .Iaddend.concentrated light energy and after the
production of the particular fluorescence .Iadd.and the background
fluorescence.Iaddend., 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 .Iadd.particular
.Iaddend.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 .[.analysing
the signals to enhance.]. .Iadd.distinguishing .Iaddend.the portion
of the signals representing the particular fluorescence during the
particular time .[.interval relative to.]. .Iadd.period from
.Iaddend.the portion of the signals representing the
.[.remaining.]. .Iadd.background .Iaddend.fluorescence during the
particular time .[.interval.]. .Iadd.period and for producing
signals representing substantially only the particular
fluorescence.Iaddend..
65. A fluorometer as set forth in claim 64 wherein the detecting
means includes means for scanning the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen to obtain the
production of the signals representative of the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen as a result of the
production of the burst of concentrated light energy.
66. The fluorometer of claim 65 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
68. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurements of the particular fluorescence from the
measurements of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences .Iaddend.from the specimen
including the particular fluorescence .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence from the specimen
.Iaddend.for detecting .[.the fluorescence.]. .Iadd.such
fluorescences .Iaddend.and for producing signals in accordance with
.[.the fluorescence.]. .Iadd.such detection.Iaddend.,
means coupled to the detecting means for controlling the detecting
means within a particular time period.Iadd., after the burst of the
concentrated light energy, .Iaddend.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 .Iadd.particular
.Iaddend.fluorescence .Iadd.and the background fluorescence from
the specimen .Iaddend.within the particular time period after the
production of the burst of .Iadd.the .Iaddend.concentrated light
energy, .Iadd.and .Iaddend.
data processing means responsive to the signals produced by the
detecting means .Iadd.during the particular time period
.Iaddend.for .[.analysing the signals to enhance.]. .Iadd.enhancing
.Iaddend.the portion of the signals representing the particular
fluorescence .Iadd.during the particular time period
.Iaddend.relative to the portion of the signals representing the
.[.remaining.]. .Iadd.background .Iaddend.fluorescence .Iadd.during
the particular time period.Iaddend., and
the data processing means including first means for differentiating
the signals produced by the detecting means at a number of time
points .Iadd.during the particular time period .Iaddend.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 .Iadd.during the
particular time period .Iaddend.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 .Iadd.during the
particular time period .Iaddend.for producing signals representing
.Iadd.substantially only .Iaddend.the particular fluorescence.
69. The fluorometer of claim 68 additionally including means for
producing a scanning of the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for forming signals representative of
the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from the
specimen.
70. The fluorometer of claim 69 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
72. The fluorometer of claim 69 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences.Iaddend..
73. The fluorometer of claim 72 wherein the means for detecting
includes a photosensitive array for detecting the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences
.Iaddend.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.]. .Iadd.fluorescences
.Iaddend.emanating from the specimen at the different
positions.
75. The fluorometer of claim 68 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.and with the
controlling means controlling the passage of the image of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.to the detecting
means and additionally including means for forming signals
including means for observing or recording .[.of.]. the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.from the
specimen.
76. The fluorometer of claim 75 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.from the specimen.
77. The fluorometer of claim 75 wherein the means for detecting
includes an image tube to reproduce the pattern of
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.emanating from a
plurality of spots on the specimen.
79. The fluorometer of claim 68 additionally including means
responsive to the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen for producing an image of the
.[.fluorescence and with the controlling means controlling the
detecting means with a particular time period by detector
responsive means.]. .Iadd.fluorescences .Iaddend.and additionally
including means for forming signals including means for observing
or recording .[.of.]. the .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.from the specimen.
80. The fluorometer of claim 79 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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 claim 84 wherein the electro-optic modulator
is formed by a cubic crystal of the class T.sub.d.
86. The fluorometer of claim 85 wherein the cubic crystal of the
class T.sub.d 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)
(CH.sub.2).sub.6 N.sub.4 (Hexamine or Hexamethylenetetramine)
(Na, Ca).sub.8-4 (SO.sub.4).sub.2-1 [(AlSiO.sub.4).sub.6 ]
(Hauynite)
GaP (Gallium phosphide)
Bi.sub.4 (GeO.sub.4).sub.3 (Bismuth germanate)
NaClO.sub.3 (Sodium chlorate)
BaTiO.sub.3 (Barium titanate)
SrTiO.sub.3 (Strontium titanate)
KTaO.sub.3 (Potassium tantalate) KTa.sub.x Nb.sub.1-x O.sub.3
(Potassium tantalate niobate)
88. The fluorometer of claim 68 wherein the .Iadd.particular
.Iaddend.time period .[.is relatively long and.]. has an ending
time sufficiently long to have substantially all of the
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.decayed to a low
value relative to the original value of .[.fluorescence.].
.Iadd.fluorescences.Iaddend..
89. The fluorometer of claim 68 wherein the .Iadd.particular
.Iaddend.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.].
.Iadd.background .Iaddend.fluorescence.
90. The fluorometer of claim 68 wherein the .Iadd.particular
fluorescence has a characteristic decay time and .Iaddend.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 .Iadd.the .Iaddend.added fluorescent label,
and the third means extracts from the individual time point signals
and the derivatives at the corresponding time points and the
characteristics decay time of the particular .[.fluorescence.].
.Iadd.fluorescences .Iaddend.the emission intensity representing
the particular fluorescence only and being separated from the
background emission.
91. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
between the measurement of the particular fluorescence and the
measurement of the background fluorescence.Iaddend., including
.Iadd.the particular fluorescence having a different decay time
than the background fluorescence, .Iaddend.
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 .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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
.Iadd.particular .Iaddend.time period has a beginning time and an
ending time to enhance the detection of the particular fluorescence
relative to the .[.total.]. .Iadd.background .Iaddend.fluorescence,
the beginning time occurring a first particular time after the
burst of concentrated light energy and during the production of the
particular fluorescence .Iadd.and the background fluorescence
.Iaddend.and the ending time occurring a second particular time
after the first particular time and during the production of the
particular fluorescence .Iadd.and the background fluorescence
.Iaddend.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, .Iadd.and .Iaddend.
means coupled to the detecting means for .Iadd.distinguishing the
particular decay time of the particular fluorescence during the
particular time period from the decay time of the background
fluorescence during the particular time period and for
.Iaddend.forming signals representative .Iadd.substantially only
.Iaddend.of the particular fluorescence from the specimen, and
means responsive to the .[.detection of.]. .Iadd.signals
representing substantially only .Iaddend.the particular
fluorescence from the specimen during the particular time period
for producing an image of .Iadd.substantially only .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.emanating from the
specimen at the different positions.
94. A fluorometer for measuring the .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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 .Iadd.and the background
fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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.Iadd., after the burst of the concentrated light energy,
.Iaddend.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.]. .Iadd.background .Iaddend.fluorescence,
the beginning time and the ending time occurring during the
production of the particular fluorescence .Iadd.and the background
fluorescence .Iaddend.from the specimen .[.as a result of.].
.Iadd.after .Iaddend.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 .Iadd.and the background fluorescence .Iaddend.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 .Iadd.distinguishing the
signals representing the particular fluorescence during the
particular time period from the signals representing the background
fluorescence during the particular time period and for
.Iaddend.forming signals representative .Iadd.substantially only
.Iaddend.of the particular fluorescence from the specimen during
the particular time period,
means responsive to the .Iadd.signals representing substantially
only the particular .Iaddend.fluorescence from the specimen for
producing an image of .Iadd.substantially only .Iaddend.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.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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.].
.Iadd.fluorescences .Iaddend.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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences
.Iaddend.from the specimen including the particular fluorescence
.Iadd.and the background fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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.Iadd., after the burst of the concentrated light energy,
.Iaddend.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 .Iadd.background
.Iaddend.fluorescence .[.including the particular flourescence.].,
the beginning time and ending time occurring during the production
of the particular fluorescence .Iadd.and the background
fluorescence .Iaddend.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,
.Iadd.data processing .Iaddend.means coupled to the detecting means
for .[.forming.]. .Iadd.differentiating the detected fluorescence,
and integrating the detected fluorescence, at progressive instants
of time and for processing the results of such differentiation and
integration to form .Iaddend.signals representative
.Iadd.substantially only .Iaddend.of the particular fluorescence
from the specimen, and
means responsive to the signals from the .[.detecting.]. .Iadd.data
processing .Iaddend.means during the particular time period
.Iadd.in representation of substantially only the particular
fluorescence from the specimen .Iaddend.for producing an image of
.Iadd.substantially only .Iaddend.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.].
.Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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 analysing the signals to enhance the portion of
the signals representing the particular fluorescence relative to
the portion of the signals representing the .[.remaining.].
.Iadd.background .Iaddend.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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in a
specimen .Iadd.and for distinguishing the measurement of the
particular fluorescence from the measurement of the background
fluorescence.Iaddend., 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 of 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 .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting .[.the fluorescence.]. .Iadd.such fluorescences
.Iaddend.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
.Iadd.after the burst of concentrated light energy .Iaddend.to
optimize the detection of the particular fluorescence during the
particular time period.Iadd., .Iaddend.the particular time period
having a beginning time and an ending time to enhance the detection
of the .Iadd.portion of the .Iaddend.signals representing the
particular fluorescence during the particular time period relative
to the portion of the signals representing the .[.remaining.].
.Iadd.background .Iaddend.fluorescence during the particular time
period, the beginning time and the ending time occurring during the
production of the particular fluorescence .Iadd.and the background
fluorescence .Iaddend.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 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.]. .Iadd.signals .Iaddend.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.]. .Iadd.fluorescences .Iaddend.emanating from a
plurality of spots on the specimen and means are included for
scanning the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.from
the specimen and the means for scanning includes an array control
for scanning the photosensitive array to reproduce the detected
.[.fluorescence.]. .Iadd.fluorescences .Iaddend.of the different
spots on the specimen.
108. The fluorometer of claim 106 wherein means are included for
scanning the .[.fluorescence.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.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.]. .Iadd.fluorescences .Iaddend.produced from the
specimen during the particular time period as a result of the burst
of concentrated light energy.
110. A fluorometer for measuring .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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 .Iadd.and the background fluorescence
from the specimen.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting such .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.Iadd., after the burst of the concentrated light
energy,.Iaddend.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.]. .Iadd.detection of .Iaddend.the .Iadd.background
.Iaddend.fluorescence during the particular time period .[.other
than the particular fluorescence.]., the beginning and ending times
occurring during the production of the particular fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
.[.as a result of.]. .Iadd.after .Iaddend.the burst of concentrated
.Iadd.light .Iaddend.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 .Iadd.and the background fluorescence .Iaddend.from
the specimen during the particular time period as a result of the
production of the burst of concentrated .Iadd.light
.Iaddend.energy, .Iadd.and .Iaddend.
data processing means responsive to the signals produced by the
detecting means during the particular time period for .[.analyzing
the signals to enhance.]. .Iadd.enhancing as a function of time
.Iaddend.the portion of the signals representing the particular
fluorescence during the particular time period relative to the
portion of the signals representing the .Iadd.background
.Iaddend.fluorescence during the particular time period .[.other
than particular fluorescence.]. .Iadd.and for producing signals
representing substantially only the particular fluorescence during
the particular time period.Iaddend., and
means responsive to the signals from the data processing means
.Iadd.in representation of substantially only the particular
fluorescence .Iaddend.for producing an image of the
.Iadd.particular .Iaddend.fluorescence.
111. The fluorometer of claim 110 wherein the means for detecting
includes a photodetector for detecting the .[.fluorescence.].
.Iadd.fluorescences .Iaddend.emanating from the specimen.
112. The fluorometer of claim .[.106.]. .Iadd.110 .Iaddend.wherein
the means for detecting includes an image tube to reproduce the
pattern of .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.].
.Iadd.fluorescences .Iaddend.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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences
.Iaddend.from the specimen including the particular fluorescence
.Iadd.and the background fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting such .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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
.Iadd.after the burst of the concentrated light energy .Iaddend.to
optimize the detection of the particular fluorescence wherein the
.Iadd.particular .Iaddend.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.]. .Iadd.detection of .Iaddend.the .Iadd.background
.Iaddend.fluorescence during the particular time period .[.other
than the particular fluorescence.]., the beginning and ending times
occurring during the production of the particular fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
.[.as a result of.]. .Iadd.after .Iaddend.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 .[.analysing
the signals to enhance.]. .Iadd.enhancing .Iaddend.the portion of
the signals representing the particular fluorescence during the
particular time period relative to the portion of the signals
representing the .Iadd.background .Iaddend.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 .Iadd.substantially .Iaddend.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.].
.Iadd.fluorescences 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.]. .Iadd.fluorescences .Iaddend.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 a 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 .Iadd.the fluorescence from a
specimen having a background fluorescence and .Iaddend.a particular
fluorescence emanating from particular fluorophore molecules in
.[.a.]. .Iadd.the .Iaddend.specimen .Iadd.and for distinguishing
the measurement of the particular fluorescence from the measurement
of the background fluorescence.Iaddend., 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.]. .Iadd.fluorescences
.Iaddend.from the specimen including the particular fluorescence
.Iadd.and the background fluorescence.Iaddend.,
means responsive to the .Iadd.particular .Iaddend.fluorescence
.Iadd.and the background fluorescence .Iaddend.from the specimen
for detecting such .[.fluorescence.]. .Iadd.fluorescences
.Iaddend.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.Iadd., after the burst of concentrated light energy,
.Iaddend.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 .Iadd.period .Iaddend.relative to the .[.portion of the
signals representing.]. .Iadd.detection of .Iaddend.the
.Iadd.background .Iaddend.fluorescence during the particular time
period .[.other than the particular fluorescence.]..Iadd.,
.Iaddend.the beginning and ending times occurring during the
production of the particular fluorescence .Iadd.and the background
fluorescence.Iaddend.,
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.]. .Iadd.after
.Iaddend.the production of the burst of concentrated light energy,
.Iadd.and .Iaddend.
data processing means responsive to the signals produced by the
detecting means during the particular time period for .[.analyzing
the signals to enhance.]. .Iadd.enhancing .Iaddend.the portion of
the signals representing the particular fluorescence during the
particular time period relative to the portion of the signals
representing the .Iadd.background .Iaddend.fluorescence during the
particular time period .[.other than the particular
fluorescence.]., and
the means for controlling the .[.passage of the image.].
.Iadd.detection of the fluorescences .Iaddend.constituting an
electro-optic modulator formed by a cubic crystal of the class
T.sub.d.
122. The fluorometer of claim 121 wherein the cubic crystal of the
class T.sub.d 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)
(CH.sub.2).sub.6 N.sub.4 (Hexamine or Hexamethylenetetramine)
(Na, Ca).sub.8-4 (SO.sub.4).sub.2-1 [(AlSiO.sub.4).sub.6
]](Hauynite)
GaP (Gallium phosphide)
Bi.sub.4 (GeO.sub.4).sub.3 (Bismuth germanate)
NaClO.sub.3 (Sodium chlorate)
BaTiO.sub.3 (Barium titanate)
SrTiO.sub.3 (Strontium titanate)
KTaO.sub.3 (Potassium tantalate)
KTa.sub.x Nb.sub.1-x O.sub.3 (Potassium tantalate niobate)
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to fluorometers for detecting a
particular fluorescence from a specimen. Specifically, the present
invention relates to a time gated fluorometer for forming an output
signal preferably representative of an image of the fluorescent
specimen and to a specific method to enhance the analysis of the
output signal received to thereby enhance the detection of the
particular fluorescence.
2. Description of the Prior Art
In general, prior art fluorometers all suffer from a common problem
of providing for a discrimination between the generated fluorescent
signal and the background noise. Certain types of conventional
fluorometers discriminate between the fluorescent signal and the
background noise on the basis of wave length. This type of
discrimination is generally not sufficient for many types of
fluorescent signals.
Another type of discrimination can be accomplished using a time
gated technique. In particular, these instruments are based on the
principle of permitting the observation of the fluorescence or
luminescence a short, and if desired a variable, time after the
excitation period. Time gated fluorometers therefore add an
additional level of discrimination by viewing the signal
fluorescence during an optimal time window. In the past, this
technique generally employed a fluorophore of long decay time in
order to allow the background fluorescence to decay.
The time gated technique is in general based on a phosphoroscope
invented by Becquerel in 1867. In the Becquerel instrument, the
luminescent substance is placed between two rotating discs which
are mounted on a common axis and which have sector shaped
apertures. The variable time gating is achieved by an adjustment of
the angle between a sector on one disc and a sector on the other.
Subsequent refinements of the time gating technique have been
accomplished by the use of spark discharges, oscilloscopes, Kerr
cells and supersonic cells.
The rotating disc invented by Becquerel was put into a conical
configuration for a microscope by Jones as described in U.S. Pat.
No. 2,071,408 in 1937. Other more recent improvements have used
electronic techniques. For example, Wieder, U.S. Pat. No.
4,341,957, provided for the gating of a detecting circuit
electronically and used a laser for excitation. In this way, as in
other refinements of Becquerel phosphoroscope, the gating mechanism
may be adjusted so that observation of the desired signal can be
optimized. Other prior art devices such as Mueller, U.S. Pat. No.
4,006,360, use electronic gating to distinguish between species of
differing decay time where two species are involved and one is
bound dye and the other is an unbound dye.
Two commercial instruments are currently available for the
measurement of decay time or lifetimes. Both of these instruments
utilize nanosec. flash sources (electric spark in air at reduced
pressure). One instrument puts the output of a photomultiplier tube
onto a fast ocilloscope. Provision is made to match the
experimental curve with a sum of up to 3 or 4 exponentials.
The second instrument excites the sample by repeated flashes from
the source (such as at 5 kHz) and pulses the photomultiplier at
increasingly longer times after the flash. The output is fed into a
recorder or computer and gives an intensity vs. time signal. In
addition, this instrument is supplied with software to reconvolute
the experimental curve by a well known method termed Linearized
Least Squares Reconvolution.
Both wavelength based discrimination and time based discrimination
suffer by having background fluorescence superimposed on the signal
with only an indirect means of segregating the two. In addition,
the use of dyes of long decay time effectively smears out the
desired signal over a long time period, thus making this signal
hard to extract. Dyes of long decay time have inherently low
extinction coefficients and therefore provide inefficient
excitation of the fluorescence.
SUMMARY OF THE INVENTION
The present invention is directed to a new type of fluorometer
which permits the signal from the fluorophore to be automatically
separated from the background in an improved manner to produce an
enhanced fluorescent signal. This enhancement of the fluorescent
signal occurs by a particular instrument design, by the type of
data which is collected and by a specific method used to process
this data.
In particular, in a specific embodiment, the invention is
implemented using optical time gating wherein the time gating
allows high quality optical imaging of the fluorescent source and
operates at speeds consistent with the use of common fluorescent
labels. This is accomplished using electro-optic modulators which
are made of crystals which are cubic and hence isotropic when
unstressed. Such modulators operate with large numerical apertures,
are fast and therefore are suitable for use in a gated fluorescence
microscope version of the present invention. As an example, the
modulators may be made of a cubic crystal material such as Cu Cl
(cuprous chloride). The use of such electro-optic modulators makes
possible the construction of a gated fluorescence microscope in
which the optical image is viewed directly such as in normal
microscopy.
The use of high quality optical imaging is not necessary if the
invention is used only as a fluorometer for detecting the
fluorescent signal as opposed to providing a representation of the
fluorescent image. However, even in the former case imaging is
advantageous since such imaging provides for an efficient
collection of light.
In one actual embodiment of the present invention, the fluorometer
may be considered to be a one pixel display. The present invention
provides for an enhanced optical image of a fluorescent specimen
formed from a large number of pixels. In one embodiment a single
pulse from a laser is used to excite fluorescence from a
microscopic spot on the specimen. The position of the spot on the
specimen depends upon the position of a stepping stage all of which
is sensed and under the control of control circuitry.
A microscopic objective focuses the light from the excited spot
onto a photodetector through an electro-optic modulator or an
equivalent structure such as an electronically time gated
photodetector. The electro-optic modulator is used as an optical
shutter. The electro-optic modulator is designed to have a high
numerical aperture to thereby allow the collection of more light
over a greater solid angle thereby permitting a higher resolution
in the image formation. The present invention also improves the
image quality to thereby provide a better signal to noise ratio
than other types of electro-optic modulators such as Pockels cells
or Kerr cells. In addition, the collection of more light is
benefical to improve the signal to noise ratio relative to a
photodetector positioned after the electro-optic modulator. The
electro-optic modulator opens at a time t.sub..alpha. after the
burst from the laser and closes at a time t.sub..beta.. The timing
control of the opening and closing of the electo-optic modulator
and the control of the laser are provided by the control
circuitry.
The fluorescence may be recovered by a photodetector and the
recovered information may be stored and processed to extract, from
the fluorescence signal, intensity as a function of time, and with
the information for the one illuminated spot stored for subsequent
display. The stepping stage may now move the specimen to a
different spot as directed by the control circuitry and the above
process is repeated until the specimen has been scanned as desired
to build up a complete enhanced image. If desired, the stored
information may be printed out in a numerical form as opposed to
providing for an actual image and, in such a generalized case, the
instrument of the present invention is characterized as a
fluorometer rather than the specialized designation of
fluoromicroscope.
In another embodiment of the invention, the image of a large number
of pixels is directly transmitted through the electro-optic
modulator to a photosensitive array. The array is then scanned to
form the complete image of the fluorescence from the specimen.
The advantages present in the apparatus and method of the present
invention are based on a number of physical principles which
underlie the instrument design and method of operation. In
particular, these physical principles include the excitation by a
concentrated pulse or burst of light energy provided by the laser
which is very short in duration compared to the decay time of the
fluorophore of interest. In addition, the excitation provided by
the laser produces a light pulse of sufficient energy to excite
substantially all of the fluorophore molecules so that
substantially all of the fluorescence of interest is at its peak
excitation when the burst ends. If the burst is too long, then the
fluorescence of interest starts to decay during excitation thereby
losing signal and contributing indirectly to photobleaching. If the
burst is of insufficient energy then random fluctuations in both
the fluorescence photon flux and electrical dark noise become more
important and affect measurement of the signal adversely.
The present invention also includes sensing of the fluorescence by
means of a photosensitive device gated by an electro-optic
modulator to respond promptly after the excitation pulse and
arranged to sense the total emission intensity over a large portion
of the entire time course of the particular fluorescence being
measured. The output of the photosensor is then recorded following
the excitation pulse and with this recording provided by suitable
fast analog or digital means.
The emission resulting from the excitation pulse is analyzed by a
particular method which permits the particular fluorescent signal,
which has a characteristic decay time, to be extracted from the
time dependence of the total emmission intensity.
The above described conditions assure that the maximum possible
fluorophore signal is obtained while minimizing photobleaching or
fading. In addition, the output signal from the fluorometer or
image of the fluoromicroscope of the present invention ideally
consists of signals from the fluorophore only. The degree to which
this ideal can be approached depends only upon the accuracy with
which the curve of intensity versus time for the particular
fluorescence can be sensed and recorded.
The present invention therefore provides for an apparatus and
method of enhancing the sensing of particular fluorescent data and
with such enhancement provided both by the structural components in
the system for detecting the fluorescence, and a method of analysis
of the fluorescent signal when detected.
BRIEF DESCRIPTION OF THE DRAWINGS
A clearer understanding of the present invention will be had with
reference to the following descriptions and drawings wherein:
FIG. 1 illustrates a generalized fluorescent decay curve after
excitation of a fluorescent specimen;
FIG. 2 illustrates a first embodiment of a pulsed light source;
FIG. 3 illustrates a second embodiment of a pulsed light
source;
FIG. 4 illustrates a first embodiment of a fluorometer exemplifying
instruments which may be formed as a measurement tool;
FIG. 5 illustrates a second embodiment of a fluorometer
exemplifying instruments which may be formed as a fluoromicroscope
and with a photosensitive array;
FIG. 6 illustrates a third embodiment of a fluorometer exemplifying
instruments which may be formed as a fluoromicroscope and with a
stepping stage; and
FIGS. 7 (a), (b) and (c) illustrate various alternative structures
for providing direct scanning of the specimen for producing X-Y
movement of the beam from the pulsed light source; and
FIG. 8 is a block diagram of a method of analysis of the
fluorescent signal .
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Fluorescent molecules are used as a label or tracer for a number of
reasons and in particular have been used in the medical field. In
general, the main problem of obtaining the fluorescent signal from
the fluorescent molecule is to separate or segregate the desired
signal from the unwanted background fluorescence.
In general, two different characteristics may be used to separate
the desired signal from the background. In particular, wave length
characteristics and decay time characteristics of the emitting
fluorescent molecules can be used to implement the separation
between signal and background. As shown in FIG. 1, once the
fluorescent molecules have been illuminated, such as by a laser
pulse, the decay of the fluorescence from the assembly of molecules
having a number of different decay times provides a time course of
intensity versus time of the general character shown in FIG. 1.
Using the concept of a time window described above, the
fluorescence may be observed between t.sub..alpha. and t.sub..beta.
where t.sub..alpha. and t.sub..beta. are in the general region of
the decay time for the particular label being measured. If the
fluorescent signal is observed only during this particular time
window, the signal from the label is enriched since rapidly
decaying fluorescence in the background, not associated with the
label, will not be sensed and slowly decaying fluorescence, again
not associated with the label, will be cut off after the time
window. The excited states of all of the molecules begin to decay
immediately but the long decay time or slowly decaying fluorescence
is spread out over a longer interval than the short decay time or
rapidly decaying fluorescence.
The present invention also provides for a method which may be
designated as Hybrid Laplace Transform Amplitude Analysis, or
amplitude analysis for short, for extracting a known decay
fluorescent component (.tau..sub.x) from a noise background
containing a multitude of different components (.tau..sub.1 . . .
.tau..sub.N). By this method the decay curve is analyzed by
differentiating the decay curve at N points (where N is the number
of fluorescent components in the background) and by integrating
over N intervals along the decay curve. By this particular method a
sufficient number of equations is obtained to solve uniquely the
desired particular fluorescent signal.
The method may be carried out over the entire time course of the
fluorescent decay or over a selected time window as described above
in which the signal has been enriched. Depending upon the
particular fluorescence being analyzed, one or the other of the
time periods may give the more rapid and incisive convergence to a
final value of N and the unknown particular fluorescent defined as
I.sub.x (0).
It is to be appreciated that the methods of the present invention
can be used in conjunction with well known methods of wavelength
discrimination.
Given a total fluorescence signal I.sub.s (t) as a function of
time, represented by a sum of unknown signals as well as the
desired signal ##EQU1## We need to evaluate I.sub.x (0). Notice
that we know I.sub.s (t) for all time. We known the characteristic
decay time (T.sub..chi. =k.sub.x.sup.-1) of the fluorescent label.
We DO NOT known I.sub.i (0), k.sub.i and wish to evaluate I.sub.x
(0). Hence we have (2N+1) unknowns. This requires (2N+1) equations
for a unique solution. We obtain one equation from equation (2) at
t=0 namely ##EQU2## By differentiating equation (2) at N points and
by integrating equation (2) over N intervals we can solve for
I.sub.x (0). Consider, for example, just one background noise term,
say,
Applying the above technique, we get
Differentiating equation (4) with respect to t we get:
Integrating equation (4) from 0 to infinity we obtain: ##EQU3##
Evaluate equation (4) and (5) at t=0 to give:
Substituting limits into equation (6) ##EQU4## We now have three
equations, (7), (8) and (9), in three unknowns, I.sub.x (0),
I.sub.t (0) and k.sub.1. Next, eliminate I.sub.1 (0) by multiplying
equation (7) by k.sub.1 and adding to equation (8), dropping (0)'s
to give equation (12)
Solving equation (12) for k.sub.1 gives: ##EQU5## Substitute this
value of k.sub.1 from equation (13) into equation (9): ##EQU6## For
I.sub.1 substitute the value from equation (7) giving: ##EQU7##
Rearranging and solving for I.sub.x gives finally ##EQU8##
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 I.sub.s
observed from the entire sample is equation (1) above. The function
I.sub.s (t) may be measured directly after a single excitation
pulse or it may be deduced from several values of ##EQU9## which is
evaluated by an integrating detector after each of several
excitation pulses.
Since the decay of all components in the fluorescence is assumed to
be exponential, zero time is arbitrary and may be taken at any
point along the curve shown in FIG. 1. Therefore any zero time
associated with equation (2) results in equation (3) as shown
above.
Equation (2) may be differentiated as follows: ##EQU10## Evaluation
of Equation (17) at N different points t.sub.1 to t.sub.N gives N
independent equations. Integration of equation (2) over N different
intervals gives an additional N equations, for example:
##EQU11##
The 2N+1 equations are sufficient to solve for I.sub.x (0) by
eliminating the N different k's and the N different I.sub.i (0)'s.
A numerical solution for I.sub.x (0) is accomplished 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 process is stopped and that
value of I.sub.x (0) 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 a sample which is stained or
to be stained. In the usual type of background measurement, the
blank or unlabeled sample must be identical in both size and
fluorescence characteristics to the labeled sample except that no
added label is present. In the NBA method the only requirement is
that the background be measured on the same type of material but
the amount need not be the same. This feature is of particular
importance, e.g., in measuring the total amount of label taken up
by a sample of tissue or by a group of cells in the field of a
fluorescence microscope. In such a situation, it is not feasible to
prepare a blank identical in size to the stained sample and unless
the specimen is fixed it is not feasible to measure before and
after staining.
The NBA method may be developed as follows. As before, the
background itensity, I.sub.b, is assumed to consist of a sum of
intensities with exponential decays: ##EQU12##
The emission from the stained sample is then of the form:
##EQU13##
The factor, .alpha., 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
Differentiating gives
Eliminating .alpha. from equations (21) and (22) gives:
Since
Substituting equation (25) into equation (23) and solving for
I.sub.x gives: ##EQU14## true for all t.
FIG. 2 illustrates a first embodiment of a pulsed light source for
use with the various fluormeters formed as embodiments of the
present invention. In FIG. 2 a laser 1 directs light energy to a
beam splitter 2. A first portion of the energy from the beam
splitter is directed to a photodetector 3 and a second portion is
directed to a first prism 4. The photo detector 3 produces an
output signal when the laser is on and such signal is coupled to
control circuitry 5.
Control signals are also applied to the control circuitry 5 so that
the laser 1 may be controlled "on" and "off" to produce a burst of
light energy of a desired short duration as described above. A
second prism 6 is positioned to receive light energy from the first
prism 4 and to redirect the light energy back to the first prism 4.
The distance between the prisms 4 and 6 may be varied to provide a
variable optical time delay so that the burst energy is in proper
phase with an optical shutter and/or detector which would be part
of a complete instrument.
FIG. 3 illustrates a second embodiment of a pulsed light source for
use with the various flurometers formed as embodiments of the
present invention. In FIG. 3, a continuous wave or wide pulse
source 7 of light directs light energy to a modulator 8. The light
source is nearly collimated, monochromatic and polarized and is
thereby similar to the output from a laser. The modulator 8 forms
an optical shutter under the control of control circuitry 9 to
produce a short burst of high energy exciting light from the
modulator 8. The modulator 8 may be formed by an electro-optic
modulator or an acousto-optic (AO) modulator.
FIG. 4 is a first embodiment of a fluorometer forming a measurement
tool. In FIG. 4 a specimen 10 to be analysed is positioned on a
surface 12. A pulsed light source 14, which may be either the light
source formed by the embodiment of FIG. 2 or the embodiment formed
by the embodiment of FIG. 3 directs a burst of exciting light to
the specimen 10.
The light energy from the light source 14 excites fluorescence in
the specimen. The excited fluorescence emits energy which is
directed to an electro-optic modulator 26 so as to produce a time
gating of the emitted flourescence. The timing control may be
provided from a control signal from the control circuitry 5 or 9 of
the pulsed light sources shown in FIG. 2 and 3. The electro-optic
modulator 26 is controlled to open at a time t.sub..alpha. after
the burst from the light source and to close at a time
t.sub..beta., as shown in FIG. 1. The emitted fluorescence is
therefore directed to a photomultiplier and digitizer 27 to detect
and digitize the output emitted fluorescence only between the times
t.sub..alpha. and t.sub..beta..
The output from the photomultiplier and digitizer 27 is then
coupled to a signal processing and display unit 29 to analyze the
information in accordance with the methods described above and to
display the results of this analysis. It is to be appreciated that
the methods of analysis may be used with a short period time gated
fluorescence output but may also be used for analysis over a longer
time period. Also, the electro-optic modulator 26 could be
eliminated and the emitted fluorescence from the specimen 10 could
be directly applied to the photomultiplier and digitizer 27 if the
photomultiplier is gated by electronic means.
FIG. 5 illustrates a first embodiment of a fluorescence microscope
exemplifying instruments which may be formed by incorporating an
electro-optic modulator and which may incorporate the methods of
analysis described above. As shown in FIG. 5, the specimen 10 to be
analyzed is positioned on the stationary surface 12. A pulsed light
source 14 is controlled to direct a pulse or burst of concentrated
light energy toward a dichroic mirror 16. The mirror 16 directs the
light energy through an objective lens 18 to the specimen 10. Light
filters may be added in the excitation and emission beams to
thoroughly isolate the fluorescence emission and to limit
excitation to a single or narrow band of wavelengths.
The light energy from the source 14 excites fluorescence in the
specimen. The excited fluorescence thereby produces a fluorescent
pattern on the specimen 10. The objective lens 18 forms an image of
the fluorescent specimen at a photo sensitive array 20 beginning at
the time t.sub..alpha. after the pulse from the source 14. This
time t.sub..alpha. is determined by control circuitry forming part
of a timing control and data processing module 22. The control
circuitry actually controls a power supply 24 which in turn
controls the operation of the electro-optic modulator 26. The
electro-optic modulator is therefore opened at the time
t.sub..alpha. to allow the image of the fluorescent specimen to be
passed to the photo sensitive array 20.
At time t.sub..beta. the electro-optic modulator 26 closes so that
the photosensitive array has detected, at a plurality of elements
in the array, information representing the time integral of the
intensity of the fluorescence decay from time t.sub..alpha. to time
t.sub..beta. after the flash of the light source 14. It is to be
appreciated that the time interval between t.sub..alpha. to
t.sub..beta. may be a time window having a relatively short
duration as described above or may extend out over the entire time
course of the fluorescent decay. The particular time interval
chosen would be dependent upon the particular type of specimen
being observed. An array control unit 28 scans each element of the
array after the electro-optic modulator 26 closes and for each
element of the array, the array control unit 28 records the time
integral of the intensity from t.sub..alpha. to t.sub..beta..
The module 22 includes a data processing portion and this portion
stores the data and then analyzes the stored data in accordance
with the methods described above to extract the desired particular
fluorescent signal from the total intensity stored signal. The
particular fluorescent signal is then used to produce an output
indication such as a signal image on a display 30 and with this
signal image representing the desired particular fluorescent signal
from the specimen 10.
A photosensitive array or multiple detector may be used in two
distinctly different modes for fluorescence measurements. In one,
all of the elements of the array view the same point but can be
used to generate, over the time course of the signal, different
mathematical properties of the signal. These properties may be
combinations of the intensity, its derivatives or integrals. In
another mode each photosensitive element derives the same type of
information but for a different point in an image of the
fluorescent sample. In this mode, the sample may have to be excited
multiple times in order to obtain the required amount of
information to solve equation (2).
It is to be appreciated that the specific embodiment of a
fluoromicroscope illustrated in FIG. 5 is illustrative only and
that various adaptations and modifications may be made. For
example, an image tube may be used in place of the photosensitive
array and the image of the fluorescence viewed directly by an
observer or by a camera or with a direct observation by an observer
or through a camera without any intervening detectors. Also, the EO
modulator can be omitted if the photosensitive array or image tube
is gated by electronic means.
FIG. 6 illustrates a fluoromicroscope exemplifying instruments
which may be formed using a stepping stage. Portions of the system
of FIG. 6 similar to those shown in FIG. 4 and 5 are given the same
reference character. Specifically, in FIG. 6 the specimen 10 is
mounted on a stepping stage 32. The stepping stage is assumed to be
initially at a first position. The light source 14 is controlled to
produce a pulse or burst of light energy to excite fluorescence
from a single microscopic spot on the specimen 10. The light source
14 directs the light energy to the specimen 10 by reflecting the
light energy from the mirror 16 and through the lens 18. The
objective lens 18 focuses the fluorescence from the excited spot on
the specimen 10 to a photodetector 34. The actual control of the
fluorescence detected by the photodetector 34 is in accordance with
the opening and closing of the electro-optic modulator 26.
The electro-optic modulator opens at a time t.sub..alpha. after the
laser flash and closes at a time t.sub..beta.. As described above,
the time interval may be a short duration time window or may be a
period of time sufficiently long to encompass a large fraction of
the entire time course for the fluorescence decay. A module 36
provides timing control, data storage and processing. Specifically,
as in the embodiment of FIG. 5, the light source is controlled to
produce the pulse of light energy. At a predetermined period of
time after the pulse, the electro-optic modulator 26 is controlled
through the power supply to open and close and thereby act as a
shutter. The information detected by the photodetector represents
the intensity as a function of time for the one illuminated spot on
the specimen. This information is stored by the module 36.
Additionally, the module provides for processing of this stored
data from the photodetector in accordance with the methods of
analysis described above to separate the desired particular
fluorescent signal from the background fluorescence. The
information may then be displayed in the display 30 and with the
displayed information representing the information for a large
number of spots on the specimen. In particular, the stepping stage
32 is controlled to repetitively step to different spots. This
stepping is under the control of the control circuitry in the
module 36. After each step, the illumination of a spot is provided
by the light source 14 and with a subsequent extraction of the
fluorescent signal. The process is repeated until the specimen has
been scanned in a desired pattern to produce the output
display.
It is to be appreciated that the specific embodiment of
fluoromicroscope illustrated in FIG. 6 is illustrative only and
that various adaptations and modifications may be made. For
example, the EO modulator may be omitted if the photodetector is
gated by electronic means.
In FIGS. 5 and 6, the information at a plurality of spots on the
specimen is detected using two different techniques. FIGS. 7 (a),
(b) and (c) illustrate alternative methods of producing this
detection of information at a plurality of spots and with these
alternate methods incorporated in a structure such as the
fluorometer of FIG. 4. Specifically, as shown in FIG. 4 at the
position of the dotted block 60, an X-Y positioner may be used to
control the exciting light from pulsed light source to excite the
specimen 10 at a plurality of spots for detection.
FIG. 7 (a) illustrates a first embodiment of the scanner 60
incorporating a pair of tilting or rotating mirrors 62 and 64 such
as galvanometer scanning mirrors each producing one axis of
movement of the exciting light from the pulsed light source 14 to
produce the X-Y scanning of the specimen 10.
FIG. 7 (b) illustrates a second embodiment of the scanner 60
incorporating an acoustic-optic (AO) modulator 66 and a tilting or
rotating mirror 68 each producing one axis of movement of the
exciting light from the pulsed light source 14 to produce the X-Y
scanning of the specimen 10.
FIG. 7 (c) illustrates a third embodiment of the scanner 60
incorporating a pair of AO modulators 70 and 72, each producing one
axis of movement of the exciting light from the pulse of light
source 14 to produce the X-Y scanning of the specimen 10.
In these scanning systems, the scanning is accomplished by movement
of the beam in the x and in the y directions so as to systemically
illuminate in succession each point in a field. If large areas are
to be scanned these systems may be combined with the stepping stage
to provide movement from field to field. Deflections may be
accomplished by the galvanometer scanning mirror which reflects the
beam in the desired direction or by the acoustic optics (AO)
modulator which deviates the beam into the desired direction.
The position of a galvanometer scanning mirror is controlled by the
current flow through a coil in a magnetic field. The deviation of a
beam by the AO modulator is a function of the frequency applied to
the AO material which then behaves like a diffraction grating.
Undeviated light is blanked off optically. In the AO material,
standing waves are set up producing a set of bands of refractive
index gradients by which the light is deviated. A sonic transducer
in contact with the AO material produces the periodic mechanical
stress within the AO material.
The electro-optic modulator 26 used in the embodiments of FIGS. 4,
5 and 6 is preferably a modulator of a high numerical aperture to
allow the collection and passage of as much light as possible. The
use of such an electro-optic modulator provides for an improved
signal to noise ratio for the overall system. In general, the
electro-optic modulator should have the following characteristics a
high speed which thereby implies a large electro-optic coefficient
at gigahertz frequencies together with small power consumption to
thereby permit reasonable size power supplies; large angular or
numerical aperture which is the most important requirement since
the numerical aperture of the optical system should not be less
than that of a good microscopic objective. The large numerical
aperture is therefore desirable to a obtain the needed optical
resolution to permit formation of a high quality optical image.
It has been generally known that crystals of the cubic class
T.sub.d (or 43 m) offer the maximum angular aperture for devices
based on longitudinal or transverse Pockels effects. The following
factors are generally involved in the choice of the particular
material to be used in the electro-optic modulator of the present
invention. Specifically, when an electric field is applied to a
cubic crystal (isotropic) of a class T.sub.d the crystal becomes
birefrigent. In general, it becomes biaxial and maximum retardation
is obtained for light in 110 direction and field in the 110
direction. If the field is applied in the 111 direction the crystal
becomes uniaxial, the 111 direction being the optic axis. A light
beam passing in any direction perpendicular to the 111 direction
has a retardation .sqroot.3/2 times the maximum retardation
mentioned above. The use of the latter (transverse) mode has the
advantage that the electrodes on the modulator need not be
transparent thereby allowing low resistivity to be easily
obtainable. The following group of cubic crystals belong to a group
from which the electro-optic modulator of the present invention may
be constructed. These cubic crystals include:
CuCl (Cuprous chloride)
CuBr (Cuprous bromide)
CuI (Cuprous iodide)
ZnS (Zinc sulfide)
ZnSe (Zinc selenide)
ZnTe (Zinc telluride)
(CH.sub.2).sub.6 N.sub.4 (Hexamine or Hexamethylenetetramine)
(Na, Ca).sub.8-4 (SO.sub.4).sub.2-1 [(AlSiO.sub.4).sub.6
](Hauynite)
GaP (Gallium phosphide)
Bi.sub.4 (GeO.sub.4).sub.3 (Bismuth germanate)
NaClO.sub.3 (Sodium chlorate)
BaTiO.sub.3 (Barium titanate)
SrTiO.sub.3 (Strontium titanate)
KTaO.sub.3 (Potassium tantalate)
KTa.sub.x Nb.sub.1-x O.sub.3 (Potassium tantalate niobate)
The use of electro-optic modulators, formed by cubic crystals of
the class T.sub.d providing for the time gating, allow for a high
quality optical imaging of the fluorescent source from the
specimens. These modulators operate with large numerical apertures
and are therefore suitable for use in the gated fluorescent
microscope of the present invention. The use of these electro-optic
modulators makes possible the production of an optical image that
can be viewed in a similar way to normal microscopy.
As disclosed above, these electro-optic modulators may be
incorporated in the two embodiments of a fluoromicroscope shown in
FIGS. 5 and 6. In addition, the output fluorescent signal may be
enhanced using the unique methods of analysis of the fluorescent
data to further separate this data from the background
fluorescence. Specifically, as shown in FIG. 8 one of the methods
extracts a particular desired fluorescent signal having a known
decay from a background of unknown noise signals. The method
encompasses differentiating the composite signal at a predetermined
number of time points as shown in block 50 and integrating the
composite signal over a predetermined number of time intervals as
shown in block 52. A computer 54 is then used to eliminate the
unknowns using the multiple equations formed by the differentiation
and integration to thereby extract the intensity of the desired
fluorescent decay signal.
The present invention therefore provides for an apparatus and
method of producing an improved detection of fluorescent signals
and provides for discrimination between the desired fluorescent
signal and the background noise.
The present invention provides for excitation by a light pulse very
short compared to the decay time of the fluorophore. The light
pulse is also of sufficient energy to excite all, or nearly all of
the fluorophore molecules in the illuminated sample. The light
pulse may be produced by a number of different means. For example,
the invention provides for the production of a short, high energy
pulse of light by the use of a pulsed laser. In addition, the
invention may provide for the short, high energy pulse of light by
means of an intense continuous source or wide pulse source in
conjunction with an optical shutter. The shutter for example, may
be an electro-optic modulator or an AO modulator.
The fluorophore signal contained in the total observed fluorescence
may be enhanced, as compared to the background fluorescence, by
means of time gating. The time gating may be implemented by a
variety of different means. For example, an electronically gated
photomultiplier tube or other suitable photodetector may provide
the time gating. Other suitable photodetectors may include an
electronically gated image tube or an electronically gated
photosensitive array. The time gating may also be provided by an
optical shutter. For example, a Pockels cell may be used to provide
an optical shutter. In addition, other electro-optic modulators may
be used to provide a shutter and the invention specifically
provides for the use of modulators made from cubic crystals of the
class T.sub.d as optical shutters having large numerical
apertures.
The various embodiments of the invention may provide for the
detection of the fluorophore signal from a variety of different
types of detectors. For example, detectors such as
photo-multipliers, image tubes or photosensitive arrays may be used
to detect the fluorescence of interest. In addition, microscope
optics may be used to form an image of the fluorescence sample with
such microscope optics forming part of the detector.
Once the fluorescence of interest is detected, the signal may be
electronically processed in a variety of different ways. In
particular, a photosensitive array may be electronically scanned so
as to determine for each pixel the fluorescence intensity averaged
over the duration of an arbitrary time window. Another processing
technique would be measurement of the fluorescence from a sample so
as to determine from each pixel the fluorescence intensity averaged
over the duration of an arbitrary time window, or as a function of
time over a time window. In the processing, the fluorescence
intensity may be digitized with any of the processing techniques.
Other aspects of the processing could be the measurement of the
fluorescence intensity as a function of time after a single
excitation pulse, or the measurement of the time integral of the
fluorescence intensity after each of a number of excitation pulses,
and with the integration carried out over a different time interval
for each pulse.
After the fluorescence of interest has been detected and processed,
it may now be analyzed using one of the methods of the present
invention. Specifically, the fluorophore signal may be extracted
from the total observed fluorescence by means of Hybrid Laplace
Transform Amplitude Analysis or by Normalized Background Analysis.
In addition, a reconstruction of an image of the fluorescence
sample may be produced from the digital data relating to the
fluorescence intensity.
Although the invention has been described with reference to
particular embodiments, it is to be appreciated that variation
adaptations and modifications may be made and the invention is only
to be limited to the appended claims.
* * * * *