Systems and methods providing efficient detection of back-scattered illumination in modulation transfer microscopy or micro-spectroscopy

1. A microscopy or micro-spectroscopy system comprising:

a first light source for providing a first illumination field at a first optical frequency ω 1 ;

a second light source for providing a second illumination field at a second optical frequency ω 2 ;

a modulator for modulating a property of the second illumination field at a modulation frequency f of at least 100 kHz to provide a modulated second illumination field;

an optical assembly including focusing optics and an optical detector system,

said focusing optics for directing and focusing the first illumination field and the modulated second illumination field through an objective lens toward the common focal volume along an excitation path; and

said optical detector system including at least one optical detector for detecting a detected first field intensity of the first illumination field that is back-scattered within a sample, wherein said optical detector provides an electrical signal representative of the detected first field intensity, and said optical detector is located proximate a portion of the excitation path; and

a processor for detecting a modulation at the frequency f of the electrical signal due to non-linear optical interaction within the common focal volume.

2. The system as claimed in claim 1 , wherein said optical detector system substantially surrounds a portion of the excitation path.

3. The system as claimed in claim 1 , wherein said processor provides an output signal that is representative of one of a gain or loss of illumination at the first optical frequency ω 1 due to the non-linear optical interaction within the common focal volume.

4. The system as claimed in claim 1 , wherein illumination at a difference frequency between ω 1 and ω 2 is resonant with a molecular vibrational frequency of the sample in the focal volume.

5. The system as claimed in claim 1 , wherein one of said first and second illumination fields is a broadband illumination field.

6. The system as claimed in claim 1 , wherein one of the first illumination field or the second illumination field is resonant with an electronic absorption of the sample.

7. The system as claimed in claim 6 , wherein the second illumination field excites molecules in the sample into an excited state.

8. The system as claimed in claim 6 , wherein the second illumination field removes molecules from a ground state by promoting them to an excited state.

9. The system as claimed in claim 1 , wherein a sum frequency of the first illumination field and the second illumination field is resonant with a two-photon electronic absorption of the sample.

10. The system as claimed in claim 1 , wherein said optical detector system includes a photo-diode having a central aperture through which the excitation path extends.

11. The system as claimed in claim 1 , wherein said optical detector system includes a plurality of photo-diodes that collectively surround the excitation path.

12. The system as claimed in claim 1 , wherein said processor provides a pixel signal that is representative of the modulation at the frequency f of the electrical signal due to non-linear optical interaction within the common focal volume as a pixel for an image for an imaging system.

13. The system as claimed in claim 1 , wherein said system includes an optical fiber for providing the first illumination field and the second illumination field to the optical assembly.

14. The system as claimed in claim 1 , wherein said optical detector system substantially surrounds the focusing optics.

15. The system as claimed in claim 1 , wherein said optical detector system substantially is positioned between the focusing optics and the sample, and surrounds a focal path of the focusing optics.

16. The system as claimed in claim 1 , wherein said focusing optics includes a gradient index (GRIN) lens.

17. The system as claimed in claim 1 , wherein said optical assembly includes a filter for passing to the optical detector only illumination from within the sample having the first optical frequency.

18. The system as claimed in claim 1 , wherein said optical assembly includes a cover that is transparent to the first optical frequency and the second optical frequency to provide a sealed optical assembly.

19. The system as claimed in claim 1 , wherein said optical assembly includes a light baffle for preventing illumination from contacting the optical detector that has not passed through an optical filter.

20. The system as claimed in claim 1 , wherein said optical assembly includes a scanning assembly for scanning the focal area within a sample in at least one direction.

21. The system as claimed in claim 20 , wherein said scanning assembly includes an electro-optical switch.

22. The system as claimed in claim 20 , wherein said scanning assembly includes at least two positionable mirrors for positioning the focal area within a range of x and y directions within the sample.

23. The system as claimed in claim 20 , wherein said scanning assembly includes a micro-electro-mechanical element.

24. The system as claimed in claim 23 , wherein said micro-electro-mechanical element provides that the focal area may be positioned within a range of x and y directions within the sample.

25. The system as claimed in claim 20 , wherein said scanning assembly includes a piezo tube actuator for positioning a tip of an optical fiber that delivers the first illumination field and the modulated second illumination field to the focal area within a range of x and y directions within the sample.

26. A method of performing microscopy or micro-spectroscopy, said method comprising the steps of:

providing a first illumination field at a first optical frequency ω 1 ;

providing a second illumination field at a second optical frequency ω 2 ;

modulating a property of the second illumination field at a modulation frequency f of at least 100 kHz to provide a modulated second illumination field;

directing and focusing the first illumination field and the modulated second illumination field through an objective lens toward the common focal volume along an excitation path;

detecting a detected first field intensity of the first illumination field that is back-scattered within a sample at an optical detector system that includes at least one optical detector, wherein said optical detector is positioned proximate a portion of the excitation path;

providing an electrical signal representative of the detected first field intensity; and

processing the electrical signal to detect a modulation at the frequency if of the electrical signal due to non-linear optical interaction within the common focal volume.

27. The method as claimed in claim 26 , wherein said optical detector system substantially surrounds a portion of the excitation path.

28. The method as claimed in claim 26 wherein said method further includes the step of providing an output signal that is representative of one of a gain or loss of illumination at the first optical frequency ω 1 due to the non-linear optical interaction within the common focal volume.

29. The method as claimed in claim 26 , wherein illumination at a difference frequency between ω 1 and ω 2 is resonant with a molecular vibrational frequency of the sample in the focal volume.

30. The method as claimed in claim 26 , wherein one of said first and second illumination fields is a broadband illumination field.

31. The method as claimed in claim 26 , wherein one of the first illumination field or the second illumination field is resonant with an electronic absorption of the sample.

32. The method as claimed in claim 31 , wherein the second illumination field excites molecules in the sample into an excited state.

33. The method as claimed in claim 31 , wherein the second illumination field removes molecules from a ground state by promoting them to an excited state.

34. The method as claimed in claim 26 , wherein a sum frequency of the first illumination field and the second illumination field is resonant with a two-photon electronic absorption of the sample.

35. The method as claimed in claim 26 , wherein said method further includes the step of providing a pixel signal that is representative of the modulation at the frequency f of the electrical signal due to non-linear optical interaction within the common focal volume as a pixel for an image for an imaging system.

36. The method as claimed in claim 26 , wherein said method further includes the step of filtering illumination from within the sample to pass to the optical detector only illumination having the first optical frequency.

37. The method as claimed in claim 26 , wherein said method further includes the step of scanning the focal area within a sample in at least one direction.

38. The method as claimed in claim 37 , wherein said step of scanning includes the step of actuating an electro-optical switch.

39. The method as claimed in claim 37 , wherein said step of scanning includes the step of actuating at least two positionable mirrors for positioning the focal area within a range of x and y directions within the sample.

40. The method as claimed in claim 37 , wherein said step of scanning includes the step of actuating a micro-electro-mechanical element.

41. The method as claimed in claim 40 , wherein said micro-electro-mechanical element provides that the focal area may be positioned within a range of x and y directions within the sample.

42. The method as claimed in claim 26 , wherein said step of scanning includes the step of actuating a piezo tube actuator for positioning a tip of an optical fiber that delivers the first illumination field and the modulated second illumination field to the focal area within a range of x and y directions within the sample.