Flow cytometer

• US 5,367,474 A
• Filed: 02/08/1993
• Issued: 11/22/1994
• Est. Priority Date: 02/08/1993
• Status: Expired due to Term

• Current Assignee: Coulter International Corp. (Danaher Corporation)
• Original Assignee: Coulter Corporation (Danaher Corporation)
• Inventors: Weber, Bruce M., Auer, Robert E., Starling, John D., Wood, James C. S.
• Primary Examiner(s): Harvey, Jack B.
• Assistant Examiner(s): MILLER, CRAIG S
• Application Number:
  US08/014,701
• Time in Patent Office: 652 Days
• Field of Search: 209/576, 250/461.2, 356/39, 356/317, 356/318, 356/442, 364/555, 364/139, 364/177, 364/413.08, 340/825.06, 340/825.07, 340/825.08
• US Class Current: 702/21
• CPC Class Codes: G01J 2001/4406 Plural ranges in circuit, e.g. switchable ranges; Adjusting sensitivity selecting gain values
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals

Abstract

A flow cytometer includes a plurality of detectors for providing voltage pulse signals over a four decade range as particles pass through an illuminated detection station. Each voltage pulse signal is processed and provided to a first sample and hold for storage. Thereafter, the value in the first sample and hold is amplified by one or thirty-two, depending upon its magnitude, and stored in a second sample and hold for subsequent provision to a sixteen bit analog to digital convertor (ADC), one signal at a time. The first sample and hold is then free to store a new value while the old value is awaiting provision to the ADC. The ADC converts the signal received from the second sample and hold to a fifteen bit digital signal and uses the sixteenth bit to manifest whether the signal stored in the second sample and hold was amplified by thirty-two.

Claims

1. A flow cytometer for detecting and identifying particles in a fluid, said flow cytometer having means for moving said particles to a detection station, means for directing energy at said detection station so that said particles intersect said energy when at said detection station, and a plurality of detectors for providing analog electric signals manifesting different results of each particle intersecting said energy, said flow cytometer comprising:

a plurality of analog signal processors, each responsive to only one detector signal for processing, delaying and storing analog data manifesting the signal provided thereto;

first controller means responsive to a trigger signal from a selected one of said analog signal processors for determining that a particle is intersecting said energy, said trigger signal being time related to the signals from said detectors, said first controller means providing signals to each of said analog signal processors for causing the storage of a delayed version of the signal applied thereto;

second controller means for polling each of said plurality of analog signal processors to receive and digitize said delayed signal stored thereby and to further process said received and digitized signals in order to identify said detected particle and each analog signal processor stores a signal related to the signal provided thereto, and provides a data signal to said second controller means in response to a poll signal therefrom, each analog processor including amplifying means for amplifying each stored signal to provide said data signal, said amplifying means amplifying said stored signal with one of a first gain or a second gain, said second gain being greater than said first gain, said amplifying means amplifying said stored signal with said second gain unless the signal value amplified with said second gain exceeds a certain value.

2. The flow cytometer according to claim 1 wherein each of said analog signal processors has first and second analog signal storage means, said first storage means storing data related to said delayed signal and said second storage means storing data related to the data stored by said first storage means during the time said second controller means polls each analog signal processor.

3. The flow cytometer according to claim 2 wherein said first storage means is enabled to store data from a newly detected particle after the data previously stored thereby has been stored in said second storage means.

4. The flow cytometer according to claim 1 wherein each analog signal processor provides an associated control signal to said second controller means manifesting the one gain of said amplifying means for the data signal being provided.

5. The flow cytometer according to claim 4 wherein said second controller means includes an analog to digital converter for converting the analog signal provided thereto to a digital signal, said second controller means digital signal being limited to N bits, at least one of which is said control signal and the remaining ones of which contain the digital signal from said analog to digital converter.

6. The flow cytometer according to claim 5 wherein each of said analog signal processors has first and second analog signal storage means, said first storage means storing data related to said delayed signal and said second storage means storing data related to the data stored by said first storage means during the time said second controller means polls each analog signal processor.

7. The flow cytometer according to claim 6 wherein said signal provided for storage to said second storage means is amplified by said amplified means.

8. The flow cytometer according to claim 1 wherein each of said analog signal processors has first and second analog signal storage means, said first storage means storing data related to said delayed signal and said second storage means storing data related to the data stored by said first storage means during the time said second controller means polls each analog signal processor.

9. The flow cytometer according to claim 8 wherein said signal provided for storage to said second storage means is amplified by said amplified means.

10. A flow cytometer for detecting particles in a fluid and determining information regarding said particles, said flow cytometer having means for moving said particles to a detection station, means for directing energy at said detection station so that said particles intersect said energy when at said detection station, and detector means for providing an electric signal manifesting the result of each particle intersecting said energy, said flow cytometer further having a detection circuit comprising:

means for detecting the presence of a particle in said detection station and providing an electric signal manifesting a parameter of said detected particle;

first storage means for storing an electric signal related to said detector means signal after said means for detecting has detected a particle in said detection station;

second storage means for storing, on command, an electric signal related to the signal stored by said first storage means;

processor means responsive to the electric signal stored by said second storage means for processing the electric signal stored by said second storage means to obtain the information regarding each particle;

command means for enabling said second storage means to store said electric signal related to the signal stored by said first storage means after said processor means has completed processing the electric signal previously stored by said second storage means and said means for detecting has detected the presence of another particle in said detection station, said command means further resets said first storage means after enabling said second storage means to store said electric signal related to the signal stored by said first storage means and said command means detects the time of occurrence of a particle entering said detection station in response to said detector means signal applied thereto; and

said flow cytometer further includes delay means for delaying said detector means signal by a delay time prior to said first storage means.

11. The flow cytometer according to claim 10 wherein said delay means signal is processed and provided to said first storage means at a first time, less than said delay time, after detecting the time of occurrence of a particle entering said detection station.

12. The flow cytometer according to claim 11 wherein said command means detects the time of occurrence of a particle leaving said detection station in response to said detector means signal applied thereto, and enables said first storage means to store said processed delay means signal at a second time, greater than said delay time, following the detection of said particle leaving said detection station.

13. The flow cytometer according to claim 12 wherein said delay means signal is processed such that the time domain integral is stored in said first storage means.

14. The flow cytometer according to claim 12 wherein said delay means signal is processed such that the peak value thereof is stored in said first storage means.

15. The flow cytometer according to claim 10 wherein said command means includes threshold means to which said detector means signal is provided, said threshold means providing a first signal after said detector means signal exceeds a threshold value and a second signal after said detector means signal falls below said threshold value, said first signal being provided to permit further processing of said delay means signal prior to storage in said first storage means and said second signal being provided to enable said first storage means to store said processed delay means signal.

16. The flow cytometer according to claim 15 wherein said delay means signal occurs between said first and second signals.

17. The flow cytometer according to claim 15 wherein said threshold means determines the time that said detector means signal is above said threshold, said first signal being provided at a time, related to said delay time, after said detector means signal exceeds said threshold if said time that said detector means signal is above said threshold exceeds a certain minimum time and said first signal being provided at a time related to the time said detector means signal falls below said threshold if said time that said detector means signal is above said threshold does not exceed said certain minimum time.

18. The flow cytometer according to claim 15 wherein said threshold means provides said second signal a fixed time after said detector means signal falls below said threshold, said delay means signal occurring between said first and second signals.

19. The flow cytometer according to claim 18 wherein said delay means signal is processed such that the time domain integral is stored in said first storage means.

20. The flow cytometer according to claim 18 wherein said delay means signal is processed such that the peak value thereof is stored in said first storage means.

21. The flow cytometer according to claim 10, wherein said processor means includes a plurality of analog signal processors and each analog signal processor stores a signal related to the signal provided thereto, and provides a data signal to said second controller means in response to a poll signal therefrom, each analog processor including amplifying means for amplifying each stored signal to provide said data signal, said amplifying means amplifying said stored signal with one of a first gain or a second gain, said second gain being greater than said first gain, said amplifying means amplifying said stored signal with said second gain unless the signal value amplified with said second gain exceeds a certain value.

22. The flow cytometer according to claim 21 wherein each analog signal processor provides an associated control signal to said second controller means manifesting the one gain of said amplifying means for the data signal being provided.

23. The flow cytometer according to claim 22 wherein said second controller means includes an analog to digital converter for converting the analog signal provided thereto to a digital signal, said second controller means digital signal being limited to N bits, at least one of which is said control signal and the remaining ones of which contain the digital signal from said analog to digital converter.

24. The flow cytometer according to claim 23 wherein each of said analog signal processors has first and second analog signal storage means, said first storage means storing data related to said delayed signal and said second storage means storing data related to the data stored by said first storage means during the time said second controller means polls each analog signal processor.

25. The flow cytometer according to claim 24 wherein said signal provided for storage to said second storage means is amplified by said amplified means.

26. The flow cytometer according to claim 21 wherein each of said analog signal processors has first and second analog signal storage means, said first storage means storing data related to said delayed signal and said second storage means storing data related to the data stored by said first storage means during the time said second controller means polls each analog signal processor.

27. The flow cytometer according to claim 26 wherein said signal provided for storage to said second storage means is amplified by said amplified means.

28. A flow cytometer for detecting and identifying particles in a fluid, said flow cytometer having means for moving said particles to a detection station means for directing energy at said detection station so that said particles intersect said energy when at said detection station, and a plurality of detectors for providing analog electric signals manifesting different results of each particle intersecting said energy, said flow cytometer comprising:

a plurality of analog signal processors, each responsive to one detector signal, each analog signal processor having delay means for providing a delayed signal, which is delayed version of that one detector means signal, first storage means for storing a processed version of said delayed signal, and second storage means for storing, on command, an electric signal related to the signal stored by said first storage means;

first controller means responsive to the detector means signal from a selected one of said analog signal processors for determining that a particle is intersecting said energy, said first controller means providing signals to each of said analog signal processors for causing the further processing of said delayed signal and the storage in said first storage means of a value related to said processed delayed signal, and

second controller means for providing signals to each of said analog signal processors to cause a value related to the value stored in said first storage means to be stored in said second storage means, said second controller polling each of said analog signal processors to receive, digitize and further process signals related to the values stored in each of said second storage means in order to identify said detected particle, said second controller means providing signals to each of said analog signal processors to cause said value related to the value stored in said first storage means to be stored in said second storage means after completing the polling each of said analog signal processors, said first controller means being disabled from determining that a particle is intersecting said energy until said second controller means provides said signals to each of said analog signal processors causing said value related to the value stored in said first storage means to be stored in said second storage means.

29. The flow cytometer according to claim 28 wherein said first controller means is enabled to detect a detector means signal during the time said second controller means is polling each of said analog signal processors.

30. The flow cytometer according to claim 29 wherein the further processing of said delayed signal is determining the time domain integral thereof.

31. The flow cytometer according to claim 30 wherein the further processing of said delayed signal is determining the maximum value thereof.

32. The flow cytometer according to claim 29 wherein said second controller means provides signals to each of said analog signal processors to cause said value related to the value stored in said first storage means to be stored in said second storage means after completing the polling each of said analog signal processors, said first controller means being disabled from determining that a particle is intersecting said energy until said second controller means provides said signals to each of said analog signal processors causing said value related to the value stored in said first storage means to be stored in said second storage means.

33. The flow cytometer according to claim 28 wherein said first controller means includes threshold detector means for determining when and how long said detector means signal from said selected one of said analog signal processors exceeds a threshold value.

34. An apparatus for detecting particles said apparatus having means for moving said particles to a detection station, means for directing energy at said detection station so that said particles intersect said energy when at said detection station, and detector means for providing an analog electric signals manifesting a result of each particle intersecting said energy, said detector means signals varying in magnitude over a certain range, said particle detecting apparatus further having converter means for converting an analog electric signal applied to an input thereof to a digital signal, said convertor means having a certain number, n, of digital outputs for manifesting the digital value of the analog signal provided to said converter means input, the improvement comprising:

first and second amplifiers each responsive to said detector means signal, said first amplifier having a first gain and said second amplifier having a second gain, said second gain being greater than said first gain;

switch means having a pair of inputs, each connected to a different one of said first and second amplifiers, and an output switchably connected with one of said inputs in response to a switch signal provided to said switch means, a switch means output signal appearing at said output;

means for determining whether said second amplifier signal exceeds a certain value and for providing said switch signal to said switch means to connect said output with said first input whenever said second amplifier signal exceeds said certain value and to connect said output with said second input whenever said second amplifier signal does not exceed said certain value, said certain value varying in magnitude with respect to time; and

said switch means output signal being coupled to said converter means input for conversion by said converter means to a digital signal manifested in n-m of said converter digital outputs, where m is at least one, said switch signal being contained in the remaining converter means digital outputs.

35. The improvement according to claim 34 wherein m equals one.

36. The improvement according to claim 34 wherein said means for determining is a comparator.

37. The improvement according to claim 36 wherein said comparator has a reference input and a data input, the output of said second amplifier means being coupled to said data input and a signal having a magnitude varying with time being coupled to said reference input.

38. The improvement according to claim 37 wherein said switch means output signal and switch signal are stored and thereafter provided to said convertor means upon request of said converter means.

39. The improvement according to claim 38 wherein m equals one.

40. The improvement according to claim 38 wherein said apparatus for detecting particles further identifies the detected particles and displays information regarding said identified particles as a histogram.

41. The improvement according to claim 34 wherein said certain value varies in magnitude with respect to time.

42. A flow cytometer for detecting and identifying various types of particles in a particle containing flowing fluid comprising:

means for causing said fluid to flow through a detection station through which is directed energy;

a plurality of detector means, each for detecting a different responses of each particle to said energy, each of said detector means providing an electric signal manifesting one response, and each electric signal having an amplitude which varies as a result of the particle type;

analog to digital converter means for responding to signals provided thereto for providing a digital signal of n bits manifesting the digital value of an analog signal provided thereto; and

a signal processor for each detector means responsive to that detector means signal for making available an analog signal for provision to said analog to digital converter means, each processor including,

first and second amplifiers each responsive to the detector means signal provided to that signal processor, said first amplifier having a first gain and said second amplifier having a second gain;

said second gain being greater than said first gain;

means for determining whether or not said second amplifier signal exceeds a certain value and for providing a switch signal manifesting that said second amplifier signal exceeds said certain value, said means for determining is an analog comparator having a reference voltage input to which a reference voltage is applied to manifest said certain value, said analog comparator further having a signal input coupled to said second input of said switch means, said analog comparator providing a signal manifesting whether or not the voltage at said signal input is greater than said reference voltage and said reference voltage is time varying between a first and second amplitude; and

switch means having a pair of inputs, each connected to a different one of said first and second amplifiers, and an output switchably connected with said first input in response to said switch signal manifesting that said second amplifier signal exceeds said certain value, and with said second input in response to said switch signal manifesting that said second amplifier signal does not exceed said certain value, a switch means output signal appearing at said output;

means for coupling said switch means output signal and said switch signal from each signal processor, one at a time, to said analog to digital converter means, said analog to digital converter means converting each coupled switch means output signal to a digital signal manifested in n-1 bits, said analog to digital converter means further including the manifestation of said switch signal as the remaining bit of each analog to digital converter means digital signal; and

means responsive to said analog to digital converter means digital signals for identifying each detected particle.

43. The flow cytometer according to claim 42 wherein said reference voltage is a sawtooth wave.

44. The flow cytometer according to claim 43 wherein means for identifying includes displaying each identified cell in a histogram format.

45. The flow cytometer according to claim 42 wherein said certain value varies with time.

46. The flow cytometer according to claim 45 wherein means for identifying includes displaying each identified cell in a histogram format.