Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

• US 8,027,032 B2
• Filed: 08/22/2008
• Issued: 09/27/2011
• Est. Priority Date: 08/22/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Freudiger, Christian, Min, Wei, Xie, Xiaoliang Sunney
• Primary Examiner(s): Geisel; Kara E
• Application Number:
  US12/196,746
• Publication Number:
  US 20100046039A1
• Time in Patent Office: 1,131 Days
• Field of Search: 356/301
• US Class Current: 356/301
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G01N 2021/653 Coherent methods [CARS]
• CPC Class Codes: G01N 2021/655 Stimulated Raman
• CPC Class Codes: G01N 2021/656 Raman microprobe
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 2201/08 Optical fibres; light guides

Abstract

A microscopy imaging system includes a first light source for providing a first train of pulses at a first center optical frequency ω₁, a second light source for providing a second train of pulses at a second center optical frequency ω₂, a modulator system, an optical detector, and a processor. The modulator system is for modulating a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses from the common focal volume by blocking the second train of pulses being modulated. The processor is for detecting, a modulation at the modulation frequency f, of the integrated intensity of the optical frequency components of the first train of pulses to provide a pixel of an image for the microscopy imaging system.

Claims

1. A microscopy imaging system comprising:

a first light source for providing a first train of pulses at a first center optical frequency ω

₁;

a second light source for providing a second train of pulses at a second center optical frequency ω

₂ such that a difference between ω

₁ and ω

₂ is resonant with a vibrational frequency of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;

a modulator system for modulating a property of the second train of pulses at a modulation frequency f of at least 100 kHz;

focusing optics for directing and focusing the first train of pulses and the second train of pulses toward a common focal volume;

an optical detector for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses transmitted or reflected through the common focal volume by blocking the second train of pulses; and

a processor for detecting a modulation at the modulation frequency f, of the integrated intensity of substantially all of the optical frequency components of the first train of pulses due to the non-linear interaction of the first train of pulses with the second train of pulses in the common focal volume, to provide a pixel of an image for the microscopy imaging system.

2. The microscopy system as claimed in claim 1, wherein said microscopy system employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provides a pump beam, and the second train of pulses that is modulated by the modulator system at the modulation frequency f provides a Stokes beam such that a Raman loss is detected at the signal processor at the modulation frequency f.

3. The microscopy system as claimed in claim 1, wherein said microscopy system employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provide a Stokes beam, and the second train of pulses that is modulated by the modulator system at the modulation frequency f provides a pump beam such that a Raman gain is detected at the signal processor at the modulation frequency f.

4. The microscopy system as claimed in claim 1, wherein said modulator provides amplitude modulation of the second train of laser pulses.

5. The microscopy system as claimed in claim 1, wherein said modulator provides polarization modulation of the second train of laser pulses.

6. The microscopy system as claimed in claim 1, wherein said modulator provides time-shift (phase) modulation of the second train of laser pulses.

7. The microscopy system as claimed in claim 1, wherein said modulator provides frequency modulation of the second train of laser pulses.

8. The microscopy system as claimed in claim 1, wherein said modulator provides double-modulation or inter-modulation of the second train of laser pulses.

9. The microscopy system as claimed in claim 1, wherein said optical detector is positioned in a forward direction with respect to the first and second trains of pulses entering the common focal volume.

10. The microscopy system as claimed in claim 1, wherein said optical detector is positioned in a reverse direction with respect to the first and second trains of pulses entering the common focal volume.

11. The microscopy system as claimed in claim 1, wherein said system further includes collecting optics for directing illumination from the focal volume toward the optical detector, and wherein the collecting optics has a numerical aperture that is at least as high as a numerical aperture of the focusing optics.

12. The microscopy system as claimed in claim 1, wherein said system further includes collecting optics for directing illumination from the focal volume toward the optical detector, and wherein the collecting optics is non-imaging optics.

13. The microscopy system as claimed in claim 1, wherein said first and second trains of pulses are directed toward the common focal volume via an optical fiber or fiber bundle.

14. The microscopy system as claimed in claim 1, wherein said first train of pulses is detected from the common focal volume via an optical fiber or fiber bundle.

15. The microscopy system as claimed in claim 1, wherein said first and second trains of pulses are directed toward the common focal volume via a first optical fiber system, and wherein said first train of pulses is detected from the common focal volume via a second optical fiber system.

16. The microscopy system as claimed in claim 1, wherein said modulation frequency f is at least 1 MHz.

17. The microscopy system as claimed in claim 1, wherein said modulator system includes any of an electro-optical modulator or an acousto-optical modulator.

18. The microscopy system as claimed in claim 1, wherein said microscopy system includes a scanning mechanism that permits one of the common focal volume and the sample to be moved in three dimensions with respect to one another such that multiple pixels of the image for the microscopy imaging system may be obtained.

19. The microscopy system as claimed in claim 1, wherein at least one of the first and second light sources includes a synchronously pumped optical parametric oscillator.

20. The microscopy system as claimed in claim 1, wherein at least one of the first and second light sources includes an electronically locked mode locked laser.

21. The microscopy system as claimed in claim 1, wherein at least one of the first and second light sources includes a broad band source from which at least two narrow bands within the broad band source band are employed.

22. The microscopy system as claimed in claim 1, wherein at least one of the first and second light sources is generated from an optical fiber that is either narrowband having a pulse width in the picosecond range, or is broadband having a pulse width in the femtosecond range.

23. The microscopy system as claimed in claim 1, wherein the modulator includes a modulated optical parametric oscillator or modulated mode-locked laser.

24. The microscopy system as claimed in claim 1, wherein the modulator includes an electronically locked mode locked laser.

25. The microscopy system as claimed in claim 1, wherein the signal processor includes an electronic passive frequency filter.

26. The microscopy system as claimed in claim 1, wherein the signal processor includes a lock-in amplifier.

27. The microscopy system as claimed in claim 1, wherein the signal processor includes a box car detector for time domain analysis.

28. The microscopy system as claimed in claim 1, wherein both the first and the second trains of pulses have a pulse width of between about 0.2 picoseconds to about 10 picoseconds.

37. The system as claimed in claim 1, wherein a modulation characteristic is at least one of amplitude or phase of the integrated intensity of substantially all of the optical frequency components of the first pulse train.

39. The microscopy system as claimed in claim 1, wherein said microscopy system includes a scanning mechanism that permits one of the common focal volume and the sample to be moved in at least one dimension with respect to one another such that multiple pixels of the image for the microscopy imaging system may be obtained.

40. The microscopy system as claimed in claim 39, wherein said microscopy system includes a scanning mechanism that permits one of the common focal volume and the sample to be moved in two dimensions with respect to one another such that multiple pixels of the image for the microscopy imaging system may be obtained.

29. A method of providing microscopy imaging, said method comprising the steps of:

providing a first train of pulses at a first center optical frequency ω

₁;

providing a second train of pulses at a second center optical frequency ω

₂ such that a difference between ω

_l and ω

₂ is resonant with a vibrational frequency of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;

modulating of a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz;

directing and focusing the first train of pulses and the second train of pulses toward a common focal volume;

detecting an integrated intensity of substantially all optical frequency components of the first train of pulses transmitted or reflected through the common focal volume by blocking the second train of pulses; and

detecting a modulation at the modulation frequency f, of the integrated intensity of the substantially all of the optical frequency components of the first train of pulses due to the non-linear interaction of the first train of pulses with the second train of pulses in the common focal volume, to provide a pixel of an image for the microscopy imaging system.

30. The method as claimed in claim 29, wherein said method employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provide a pump beam, and the second train of pulses that is modulated at the modulation frequency f provides a Stokes beam such that a Raman loss is detected at the modulation frequency f.

31. The method as claimed in claim 29, wherein said method employs stimulated Raman spectroscopy as a contrast mechanism, and wherein the first train of pulses provide a Stokes beam, and the second train of pulses that is modulated at the modulation frequency f provides a pump beam such that a Raman gain is detected at the modulation frequency f.

32. The method as claimed in claim 29, wherein said step of modulating the second train of pulses at a modulation frequency f of at least 100 kHz involves providing amplitude modulation of the second train of laser pulses.

33. The method as claimed in claim 29, wherein said step of modulating the second train of pulses at a modulation frequency f of at least 100 kHz involves providing polarization modulation of the second train of laser pulses.

34. The method as claimed in claim 29, wherein said step of modulating the second train of pulses at a modulation frequency f of at least 100 kHz involves providing phase modulation of the second train of laser pulses.

35. The method as claimed in claim 29, wherein said step of modulating the second train of pulses at a modulation frequency f of at least 100 kHz involves providing frequency modulation of the second train of laser pulses.

36. The method as claimed in claim 29, wherein said first and second trains of pulses are directed toward the common focal volume via a first optical fiber system, and wherein said first train of pulses is detected from the common focal volume via a second optical fiber system.

38. The method as claimed in claim 29, wherein a modulation characteristic is at least one of amplitude or phase of the integrated intensity of substantially all of the optical frequency components of the first pulse train.

41. The method as claimed in claim 29, wherein said method further includes the steps of:

scanning one of the common focal volume and the sample in at least one dimension with respect to one another; and

providing an image comprised of a plurality of pixels in a microscopy imaging system, wherein each pixel is associated with a scanning position.

42. The method as claimed in claim 41, wherein said step of scanning one of the common focal volume and the sample involves scanning in at least two dimensions with respect to one another.

43. The method as claimed in claim 41, wherein said step of scanning one of the common focal volume and the sample involves scanning in three dimensions with respect to one another.