SYSTEM AND METHOD FOR MONITORING CELLULAR ACTIVITY

• US 20090095919A1
• Filed: 12/15/2008
• Published: 04/16/2009
• Est. Priority Date: 07/30/1999
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Lansford, Russell D., Fraser, Scott E., Bearman, Gregory H.
• Application Number:
  US12/335,380
• Publication Number:
  US 20090095919A1
• Time in Patent Office: Days
• US Class Current: 250/459.1
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

Claims

1. A system, comprising:

a single photon laser configured to emit radiation capable of exciting a plurality of different excitable markers in a region of a cellular specimen to cause the plurality of different excitable markers to radiate florescence;

a focusing lens configured to focus radiation emitted by the single photon laser on the region of the cellular specimen;

a dichroic mirror configured to reflect the fluorescence;

a grating configured to separate the reflected fluorescence into wavelength bands of fluorescence;

a linear array of high gain photomultiplier tubes configured so that each of the wavelength bands of fluorescence is incident upon a corresponding one of the high gain photomultiplier tubes and so that each of the high gain photomultiplier tubes generates a corresponding analog signal;

a plurality of digital to analog converters, each digital to analog converter being configured to convert one of the analog signals from the linear array of high gain photomultiplier tubes to a digital signal; and

a processor configured to linearly unmix the digital signals.

2. The system of claim 1, wherein the dichroic mirror is configured so that radiation from the single photon laser that is reflected from the specimen passes through the dichroic mirror.

3. The system of claim 1, wherein the processor is configured to identify the contribution to the fluorescence from each of the plurality of excitable markers.

15. A method, comprising:

using the system of claim 1 to monitor cellular activity in a cellular specimen.

16. A method, comprising:

using the system of claim 3 to monitor cellular activity in a cellular specimen.

4. A system, comprising:

a single photon laser microscope operative to cause a plurality of different excitable markers in a region of a cellular specimen to radiate fluorescence;

photomultiplier tubes, each of the plurality of photomultiplier tubes being configured to receive a corresponding wavelength band of the florescence so that each of the photomultiplier tubes generates a corresponding analog signal;

a plurality of digital to analog converters, each digital to analog converter being configured to convert one of the analog signals from the linear array of high gain photomultiplier tubes to a digital signal; and

a processor configured to unmix the digital signals.

5. The system of claim 4, wherein the single photon laser microscope comprises a single photon laser configured to emit the radiation.

6. The system of claim 5, further comprising a focusing lens configured to focus the radiation to the region of the cellular specimen.

7. The system of claim 4, further comprising a dichroic mirror configured to reflect the fluorescence.

8. The system of claim 7, wherein the dichroic mirror is configured so that radiation from the single photon laser that is reflected from the specimen passes through the dichroic mirror.

9. The system of claim 4, further comprising a grating configured to separate the fluorescence into wavelength bands of fluorescence.

10. The system of claim 4, wherein the photomultiplier tubes form a linear array.

11. The system of claim 10, wherein the photomultiplier tubes are high gain photomultiplier tubes.

12. The system of claim 4, wherein the photomultiplier tubes are high gain photomultiplier tubes.

13. The system of claim 4, wherein the processor is configured to linearly unmix the digital signals.

14. The system of claim 4, wherein the processor is configured to identify the contribution to the fluorescence from each of the plurality of excitable markers.

17. A method, comprising:

applying a plurality of different excitable markers to a cellular specimen;

applying light to the cellular specimen from a single-photon laser microscope to cause fluorescence to be radiated from the region by markers in that region of the cellular specimen;

using a grating to separate the fluorescence into wavelength bands of fluorescence; and

using a linear array of photomultiplier tubes to detect the florescence, each photomultiplier tube receiving one of the wavelength bands of fluorescence and generating a corresponding analog signal;

converting the analog signals to digital signals; and

linearly unmixing the digital signals.

18. The method of claim 17, further comprising identifying the contribution to the fluorescence from each of the plurality of excitable markers.

19. A method, comprising:

applying light from a single-photon laser to a region of a cellular specimen having a plurality of different excitable markers, thereby causing florescence to be radiated from the region of the cellular specimen;

separating the fluorescence into wavelength bands of florescence using a grating; and

detecting the fluorescence via photomultiplier tubes, each photomultiplier tube receiving one of the wavelength bands of florescence and generating a corresponding analog signal;

converting the analog signals to digital signals; and

linearly unmixing the digital signals.

20. The method of claim 19, further comprising applying the plurality of different excitable markers to the cellular specimen.

21. The method of claim 19, wherein the single photon laser is part of a single photon laser microscope.

22. The method of claim 19, wherein the photomultiplier tubes form a linear array.

23. The method of claim 22, wherein the photomultiplier tubes are high gain photomultiplier tubes.

24. The method of claim 19, wherein the photomultiplier tubes are high gain photomultiplier tubes.

25. The method of claim 19, further comprising identifying the contribution to the fluorescence from each of the plurality of excitable markers.

26. A system, comprising:

a single photon laser configured to emit radiation capable of exciting a plurality of different excitable markers in a region of a cellular specimen to cause the plurality of different excitable markers to radiate fluorescence;

a focusing lens configured to focus radiation emitted by the single photon laser on the region of the cellular specimen;

a dichroic mirror configured to reflect the fluorescence;

a grating configured to separate the reflected fluorescence into wavelength bands of fluorescence;

a linear array of high gain photomultiplier tubes configured so that each of the wavelength bands of fluorescence is incident upon a corresponding one of the high gain photomultiplier tubes and so that each of the high gain photomultiplier tubes generates a corresponding analog signal;

a plurality of digital to analog converters, each digital to analog converter being configured to convert one of the analog signals from the linear array of high gain photomultiplier tubes to a digital signal; and

a processor configured to identify the contribution to the fluorescence from each of the plurality of excitable markers.

27. A system, comprising:

a single photon laser microscope operative to cause a plurality of different excitable markers in a region of a cellular specimen to radiate fluorescence;

photomultiplier tubes, each of the plurality of photomultiplier tubes being configured to receive a corresponding wavelength band of the fluorescence so that each of the photomultiplier tubes generates a corresponding analog signal;

a plurality of digital to analog converters, each digital to analog converter being configured to convert one of the analog signals from the linear array of high gain photomultiplier tubes to a digital signal; and

a processor configured to identify the contribution to the fluorescence from each of the plurality of excitable markers.