Time dependent fluorescence measurements

• US 20050046848A1
• Filed: 08/26/2004
• Published: 03/03/2005
• Est. Priority Date: 08/26/2003
• Status: Abandoned Application

• Current Assignee: Blueshift Biotechnologies, Inc. (Danaher Corporation)
• Original Assignee: Blueshift Biotechnologies, Inc. (Danaher Corporation)
• Inventors: Comita, Paul B., Brunfeld, Andrei, Cromwell, Evan F., Seipert, Christopher J., Adam, Johann F.
• Application Number:
  US10/927,748
• Publication Number:
  US 20050046848A1
• Time in Patent Office: Days
• US Class Current: 356/417
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0048 scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• CPC Class Codes: G02B 21/0068 arrangements using polarisation
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

Methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample. The apparatus has a light source, one or more illumination optical elements, a scanner, one or more collection optical elements, and a device forming an aperture that limits detection of light from the sample. The illumination optical elements direct a light beam from the light source onto the sample. The scanner scans the light beam across the sample. The collection optical elements collect light from the sample and transmit the collected light to a detector. None of the collection optical elements are included among the illumination optical elements. The device forming an aperture limits detection of light from the sample to light associated with a limited vertical depth within the sample, and is one of the collection optical elements.

Claims

1. An apparatus for collecting optical data pertaining to one or more characteristics of a sample, the apparatus comprising:

a light source;

one or more illumination optical elements for directing a light beam from the light source onto the sample;

a scanner for scanning the light beam across the sample;

one or more collection optical elements for collecting light from the sample and transmitting the collected light to a detector, wherein none of the one or more collection optical elements are included among the one or more illumination optical elements; and

a device forming an aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample, wherein the device is one of the collection optical elements.

2. The apparatus of claim 1, wherein the device forming the aperture is a slit aperture.

3. The apparatus of claim 1, wherein the device forming the aperture is a bundle of optical fibers.

4. The apparatus of claim 3, wherein light entering different optical fibers in the bundle of optical fibers corresponds to light at different vertical depths within the sample.

5. The apparatus of claim 1, wherein the collection optical elements collect light from a scan line on the sample with substantially uniform efficiency.

6. The apparatus of claim 5, wherein the collection optical elements include one of a cylindrical lens and a spherical lens.

7. The apparatus of claim 1, further comprising two or more detectors offset from one another with respect to a path for collecting the light from the sample, wherein each of the two or more detectors is positioned to capture light being emitted from a different vertical depth within the sample.

8. The apparatus of claim 7, further comprising logic for adjusting the position of the sample with respect to the collection optical elements in response to light intensity detected at the two or more detectors to maintain a substantially uniform vertical depth from position to position on the sample.

9. The apparatus of claim 1, wherein the detector includes at least one of:

a photomultiplier detector, a photodiode device, a charge coupled device, and a microchannel plate.

10. The apparatus of claim 1, further comprising two or more detectors for receiving light from the same region of the sample and detecting two or more different characteristics of the sample.

11. The apparatus of claim 10, wherein the two or more different characteristics include different polarizations of the light, different frequencies of the light, and different lifetimes.

12. The apparatus of claim 10, further comprising logic for examining the correlation between the signals obtained from the two or more detectors to identify objects and exclude background noise.

13. The apparatus of claim 1, further comprising logic for limiting collection of optical data to regions of the sample known or detected to hold particular objects to be characterized on the sample, the logic being implemented in computer software or computer hardware instructions that can be read and executed by a processor operatively connected to the detector.

14. The apparatus of claim 13, wherein the logic for limiting collection of optical data limits collection by only recording optical data when an intensity of the collected light is above a certain adjustable threshold value.

15. The apparatus of claim 13, wherein the logic for limiting collection of optical data limits collection by only recording optical data during time periods when the beam from the light source is scanned across an area of interest on the sample.

16. The apparatus of claim 1, wherein the light source is one of:

a continuous wave laser, a pulsed laser, a mode-locked high repetition rate laser, and a Q-switched laser.

17. The apparatus of claim 16, wherein the continuous wave laser is one of:

a diode laser, a diode-pumped solid state laser, a gas laser, and a tunable solid state laser system.

18. The apparatus of claim 16, wherein the continuous wave laser is modulated in a frequency range of 1 kilohertz to 2 Gigahertz.

19. The apparatus of claim 16, wherein the pulsed laser is configured to emit pulses in a frequency range of 1 Hertz-100 Megahertz with a spacing ranging from 10 nanoseconds to 1 second.

20. The apparatus of claim 16, wherein the mode-locked laser has a repetition rate that is higher than or equal to 10 Megahertz.

21. The apparatus of claim 16, wherein the Q-switched laser is pulsed at a frequency in the range of 1 Hertz to 1 Megahertz.

22. The apparatus of claim 1, wherein the light beam emitted from the light source is intensity modulated in time with a frequency in the range of 1 Hertz to 2 Gigahertz.

23. The apparatus of claim 1, wherein the scanner includes one or more polygonal mirrors being rotated by a scanning element to scan the light beam across the sample.

24. The apparatus of claim 1, wherein the scanner includes one or more mirrors being moved by a galvanometer to scan the light beam across the sample.

25. The apparatus of claim 1, wherein the scanner is a resonant mirror scanner.

26. The apparatus of claim 1, wherein the one or more illumination optical elements include a telecentric lens.

27. An apparatus for collecting optical data pertaining to one or more characteristics of a sample, the apparatus comprising:

a light source;

one or more illumination optical elements for directing a light beam from the light source of a first frequency onto the sample;

a scanner for scanning the light beam across the sample; and

one or more collection optical elements for collecting light of a second frequency from the sample and transmitting the light to a detector, wherein none of the one or more collection optical elements are included among the one or more illumination optical elements, and wherein the first and second frequencies are different.

28. The apparatus of claim 27, wherein the collection optical elements include one or more of a bandpass filter and a cutoff filter for determining the second frequency.

29. The apparatus of claim 27, further comprising two or more detectors offset from one another with respect to a path for collecting the light from the sample, wherein each of the two or more detectors is positioned to capture light being emitted from a different vertical depth within the sample.

30. The apparatus of claim 29, further comprising logic for adjusting the position of the sample with respect to the collection optical elements in response to light intensity detected at the two or more detectors to maintain a substantially uniform vertical depth from position to position on the sample.

31. The apparatus of claim 27, further comprising two or more detectors for receiving light from the same region of sample and detecting two or more different characteristics of the sample.

32. The apparatus of claim 31, wherein the two or more different characteristics include different polarizations of the light, different frequencies of the light, and different lifetimes.

33. The apparatus of claim 27, further comprising logic for limiting collection of optical data to regions of the sample known or detected to hold particular objects to be characterized on the sample, the logic being implemented in computer software or computer hardware instructions that can be read and executed by a processor operatively connected to the detector.

34. The apparatus of claim 27, wherein the logic for limiting collection of optical data limits collection by only recording optical data when an intensity of the collected light is above a certain adjustable threshold value and the optical data meets at least one additional criterion.

35. The apparatus of claim 27, wherein the logic for limiting collection of optical data limits collection by only recording optical data during time periods when the beam from the light source is scanned across an area of interest on the sample.

36. The apparatus of claim 27, wherein the one or more collection optical elements include a device forming an aperture that limits detection of light from the sample to light associated with a limited depth of focus within the sample.

37. The apparatus of claim 27, wherein the device forming the aperture is a slit aperture.

38. The apparatus of claim 27, wherein the device forming the aperture is a bundle of optical fibers.

39. The apparatus of claim 38, wherein light entering different optical fibers in the bundle of fibers corresponds to light at different vertical depths within the sample.

40. The apparatus of claim 27, wherein the one or more illumination optical elements include a telecentric lens.

41. An apparatus for collecting optical data pertaining to one or more characteristics of a sample, the apparatus comprising:

a light source;

one or more illumination optical elements for directing a light beam from the light source onto the sample;

a scanner for scanning the light beam across the sample;

one or more collection optical elements for collecting light from the sample and transmitting the collected light to one or more detectors; and

a first device that limits detection of light from the sample to light associated with a first vertical depth within the sample,

a second device that limits detection of light from the sample to light associated with a second, different, vertical depth within the sample,

wherein the first and second devices are collection optical elements.

42. The apparatus of claim 41, further comprising logic for automatically adjusting the vertical position of the sample with respect to the collection optical elements in response to the relative light intensity collected at the first and second devices in order to maintain a consistent vertical position of the sample with respect to the collection optical elements during scanning.

43. The apparatus of claim 41, wherein at least one of the first device and the second device is an optical fiber.

44. The apparatus of claim 43, wherein the first device comprises a first row of optical fibers and the second device comprises a second row of optical fibers.

45. The apparatus of claim 41, wherein the one or more detectors comprise one or more microchannel plates arranged to separately detect light from the first and second devices.

46. The apparatus of claim 41, wherein the one or more detectors includes at least one of:

a photomultiplier detector, a photodiode device, a microchannel plate, and a charge coupled device.

47. The apparatus of claim 41, further comprising two or more detectors for receiving light from the sample and detecting two or more different characteristics of the light from the sample.

48. The apparatus of claim 41, wherein the light beam from the light source is monochromatic having a first wavelength and wherein one or more of the collection optics and the one or more detectors is tuned to collect light at a second wavelength, which is different from the first wavelength.

49. The apparatus of claim 41, further comprising a third device that limits detection of light from the sample to light associated with a third, different, vertical depth within the sample.