Measuring time dependent fluorescence

• US 20050046849A1
• Filed: 08/26/2004
• Published: 03/03/2005
• Est. Priority Date: 08/26/2003
• Status: Active Grant

• 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.
• Granted Patent: US 7,576,862 B2
• 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. A light beam of a first frequency is scanned onto a sample surface using one or more illumination optical elements. Light of a second frequency is collected from a scan line on the sample surface using one or more collection optical elements. None of the one or more collection optical elements are included among the one or more illumination optical elements. The collected light is transmitted to a detector.

Claims

1. A method of collecting optical data pertaining to one or more characteristics of a sample, the method comprising:

scanning a light beam of a first frequency onto a sample surface using one or more illumination optical elements;

collecting light of a second frequency from a scan line on the sample surface using one or more collection optical elements, wherein none of the one or more collection optical elements are included among the one or more illumination optical elements; and

transmitting collected light to a detector.

2. The method of claim 1, wherein the first frequency is identical to the second frequency.

3. The method of claim 1, wherein collecting light includes:

collecting light through 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.

4. The method of claim 3, wherein collecting light includes:

collecting light through a slit aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample.

5. The method of claim 3, wherein collecting light includes:

collecting light using a bundle of optical fibers.

6. The method of claim 5, wherein light entering different optical fibers in the bundle of optical fibers corresponds to light at different vertical depths within the sample.

7. The method of claim 1, wherein collecting light includes:

collecting light from a scan line on the sample with substantially uniform efficiency using the one or more optical elements.

8. The method of claim 7, wherein collecting light includes:

collecting light using at least one of;

a cylindrical lens and a spherical lens.

9. The method of claim 1, wherein transmitting the collected light includes:

directing the collected light from the sample to two or more detectors offset from one another with respect to a path for collecting the light, wherein each of the two or more detectors is positioned to capture light being emitted from a different vertical depth of the sample.

10. The method of claim 9, further including:

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.

11. The method of claim 1, wherein the detector comprises at least one of:

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

12. The method of claim 1, wherein transmitting the collected light includes:

directing the collected light from the sample to two or more detectors; and

further comprising;

detecting two or more different characteristics of the light from the sample.

13. The method of claim 12, wherein detecting two or more different characteristics include:

detecting different polarizations, detecting different frequencies of light, detecting different frequencies of signal modulation, or detecting different time-gated regions.

14. The method of claim 1, further including:

automatically limiting the collection of optical data to regions of the sample known or detected to hold particular objects to be characterized on the sample.

15. The method of claim 14, wherein automatically limiting the collection of optical data includes:

recording optical data only when an intensity of the collected light is above a certain adjustable threshold value, and the optical data meets at least one additional criterion.

16. The method of claim 14, wherein automatically limiting the collection of optical data includes:

recording optical data only during time periods when the beam from the light source is scanned across an area of interest on the sample.

17. The method of claim 1, wherein scanning a light beam includes:

scanning a light beam from a light source that is one of;

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

18. The method of claim 17, wherein the pulsed laser is configured to emit pulses in a frequency range of 10-100 Megahertz with a spacing ranging from 100 picoseconds to 10 microseconds.

19. The method of claim 17, wherein the mode-locked laser has a repetition rate that is higher than or equal to 10 Megahertz.

20. The method of claim 17, wherein the Q-switched laser is pulsed at a frequency in the range of 1 Hertz to 1 Megahertz.

21. The method of claim 1, wherein scanning includes:

scanning a light beam from a light source that is intensity modulated in time with a frequency in the range of 1 Hertz to 2 Gigahertz.

22. The method of claim 1, wherein scanning includes:

scanning a light beam with a scanner that includes one or more polygonal mirrors being rotated by a scanning element to scan the light beam across the sample.

23. The method of claim 1, wherein scanning includes:

scanning a light beam with a scanner that includes one or more mirrors being moved by a galvanometer to scan the light beam across the sample.

24. The method of claim 1, wherein scanning includes:

scanning a light beam with a resonant mirror scanner.

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

26. A method of collecting optical data pertaining to one or more characteristics of a sample, the method comprising:

scanning a light beam of a first frequency onto a sample surface using one or more illumination optical elements;

collecting light of a second frequency from a scan line on the sample surface using one or more collection optical elements, wherein the light is collected through an aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample; and

transmitting the collected light to a detector.

27. The method of claim 26, wherein collecting light includes:

collecting light through a slit aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample.

28. The method of claim 27, wherein collecting light includes:

collecting light using a bundle of optical fibers.

29. The method of claim 28, wherein light entering different optical fibers in the bundle of optical fibers corresponds to light at different vertical depths within the sample.

30. The method of claim 26, wherein collecting light includes:

collecting light from a scan line on the sample with substantially uniform efficiency using the one or more optical elements.

31. The method of claim 30, wherein collecting light includes:

collecting light using one of a cylindrical lens and a spherical lens.

32. The method of claim 26, wherein transmitting the collected light includes:

directing the collected light from the sample to two or more detectors offset from one another with respect to a path for collecting the light, wherein each of the two or more detectors is positioned to capture light being emitted from a different vertical depth of the sample.

33. The method of claim 32, further including:

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.

34. The method of claim 26, wherein with the detector comprises at least one of:

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

35. The method of claim 26, wherein transmitting the collected light includes:

directing the collected light from the sample to two or more detectors; and

further comprising;

detecting two or more different characteristics of the light from the sample.

36. The method of claim 35, wherein detecting two or more different characteristics include:

detecting different polarizations, detecting different frequencies of light, detecting different frequencies of signal modulation, or detecting different time-gated regions.

37. The method of claim 26, further including:

automatically limiting the collection of optical data to regions of the sample known or detected to hold particular objects to be characterized on the sample.

38. The method of claim 37, wherein automatically limiting the collection of optical data includes:

recording optical data only when an intensity of the collected light is above a certain adjustable threshold value, and the optical data meets at least one additional criterion.

39. The method of claim 37, wherein automatically limiting the collection of optical data includes:

recording optical data only during time periods when the beam from the light source is scanned across an area of interest on the sample.

40. The method of claim 26, wherein scanning a light beam includes:

scanning a light beam from a light source that is one of;

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

41. The method of claim 40, 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.

42. The method of claim 40, wherein the mode-locked laser has a repetition rate that is higher than or equal to 10 Megahertz.

43. The method of claim 40, wherein the Q-switched laser is pulsed at a frequency in the range of 1 Hertz to 1 Megahertz.

44. The method of claim 26, wherein scanning includes:

scanning a light beam from a light source that is intensity modulated in time with a frequency in the range of 1 Hertz to 2 Gigahertz.

45. The method of claim 26, wherein scanning includes:

scanning a light beam with a scanner that includes one or more polygonal mirrors being rotated by a scanning element to scan the light beam across the sample.

46. The method of claim 26, wherein scanning includes:

scanning a light beam with a scanner that includes one or more mirrors being moved by a galvanometer to scan the light beam across the sample.

47. The method of claim 26, wherein scanning includes:

scanning a light beam with a resonant mirror scanner.

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

49. A method of collecting optical data pertaining to one or more characteristics of a sample, the method comprising:

scanning a light beam onto a sample surface using one or more illumination optical elements;

collecting light from a scan line on the sample surface using one or more collection optical elements, wherein the light is collected through (i) a first device that limits detection of light from the sample to light associated with a first vertical depth within the sample and (ii) a second device that limits detection of light from the sample to light associated with a second, different, vertical depth within the sample; and

transmitting the collected light from the first and second devices to one or more detectors.

50. The method of claim 49, further comprising 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.

51. The method of claim 49, wherein at least one of the first device and the second device is an optical fiber.

52. The method of claim 51, wherein the first device comprises a first row of optical fibers and the second device comprises a second row of optical fibers.

53. The method of claim 49, wherein the one or more detectors comprise one or more microchannel plates arranged to separately detect light from the first and second devices.

54. The method of claim 49, further comprising detecting two or more different characteristics of the light from the sample.