Fluorescence enhanced photothermal infrared spectroscopy and confocal fluorescence imaging
Embodiments disclosed include methods and apparatus for Fluorescent Enhanced Photothermal Infrared (FE-PTIR) spectroscopy and chemical imaging, which enables high sensitivity and high spatial resolution measurements of IR absorption with simultaneous confocal fluorescence imaging. In various embodiments, the FE-PTIR technique utilizes combined/simultaneous OPTIR and fluorescence imaging that provides significant improvements and benefits compared to previous work by simultaneous detection of both IR absorption and confocal fluorescence using the same optical detector at the same time.
1. A method of performing infrared imaging of a sample, the method comprising:
illuminating the sample with a beam of excitation radiation to excite autofluorescent emission from the sample;
capturing at least a portion of the autofluorescent emission from the sample with a camera-based detector to produce a frame autofluorescent image of the sample;
illuminating at least a portion of the sample corresponding to the frame autofluorescent image with a beam of infrared radiation;
capturing at least a portion of the autofluorescent emission from the sample with the camera-based detector while the portion of the autofluorescent emission from the sample is perturbed by the infrared light absorbed by the sample to produce an IR-absorbed frame autofluorescent image; and
constructing a signal indicative of infrared absorption by the sample using the frame autofluorescent image and the IR-absorbed frame autofluorescent image.
2. The method of claim 1 , further comprising capturing a plurality of frame autofluorescent images and a plurality of IR-absorbed frame autofluorescent images, wherein constructing the signal indicative of infrared absorption comprises:
determining a time averaged light level fluoresced by the region in the IR-absorbed frame autofluorescent image;
determining a time averaged light level fluoresced by the region in the frame autofluorescent image; and
comparing the time averaged light level of the IR-absorbed frame autofluorescent image with the time averaged light level of the frame autofluorescent image.
3. The method of claim 1 , wherein the camera-based detector has a frame rate of at least a few frames per second.
4. The method of claim 3 , wherein the frame rate of the camera-based detector is synchronized with a modulation rate of the beam of infrared radiation by a modulation controller.
5. The method of claim 4 , wherein the modulation controller is a lock-in controller.
6. The method of claim 1 , wherein the portion of the autofluorescent emission from the sample is perturbed by the infrared light to produce an IR-absorbed frame autofluorescent image while the beam of infrared radiation is incident on the region.
7. The method of claim 1 , wherein the autofluorescent emission excited from the sample has at least one wavelength that is shorter than the wavelength of the beam of infrared radiation.
8. The method of claim 1 , wherein the infrared source is a broadband infrared source.
9. The method of claim 1 , wherein the infrared source is a tunable infrared source having a wavelength that is tunable between 2-25 microns.
10. The method of claim 1 , wherein the excitation radiation has a wavelength that is outside of a range of the infrared light source.
11. The method of claim 1 , wherein constructing the signal is performed by at least one of a lock-in amplifier, a notch filter, an RMS-to-DC converter, a Fourier transform, and a resonant amplifier.
12. The method of claim 1 , wherein the sample comprises at least one of a biological cell, a biological tissue, and a biological organism.
13. A microscope system of performing infrared imaging of a sample, the microscope system comprising:
an excitation light source configured to illuminate the sample with a beam of excitation radiation to excite autofluorescent emission from the sample;
a camera-based detector arranged to capture at least a portion of the autofluorescent emission from the sample to produce a frame autofluorescent image of the sample;
an infrared light source configured to illuminate at least a portion of the sample corresponding to the frame autofluorescent image with a beam of infrared radiation;
a camera-based detector configured to capture at least a portion of the autofluorescent emission from the sample while the portion of the autofluorescent emission from the sample is perturbed by the infrared light absorbed by the sample to produce the IR-absorbed frame autofluorescent image; and
a processor configured to construct a signal indicative of infrared absorption by the sample using the autofluorescent frame and the autofluorescent IR-absorbed frame.
14. The system of claim 13 , further comprising capturing a plurality of autofluorescent frames and a plurality of autofluorescent IR-absorbed frames, wherein constructing the signal indicative of infrared absorption comprises:
determining a time averaged light level fluoresced by the region in the IR-absorbed frame autofluorescent image;
determining a time averaged light level fluoresced by the region in the frame autofluorescent image; and
comparing the time averaged light level of the IR-absorbed frame autofluorescent image with the time averaged light level of the frame autofluorescent image.
15. The system of claim 13 , wherein the camera-based detector has a frame rate of at least a few frames per second.
16. The system of claim 15 , wherein the frame rate of the camera-based detector is synchronized with a modulation rate of the beam of infrared radiation by a modulation controller.
17. The system of claim 16 , wherein the modulation controller is a lock-in controller.
18. The system of claim 13 , wherein the portion of the autofluorescent emission from the sample is perturbed by the infrared light absorbed by the sample to produce a IR-absorbed frame autofluorescent image while the beam of infrared radiation is incident on the region.
19. The system of claim 13 , wherein the autofluorescent emission excited from the sample has at least one wavelength that is shorter than the wavelength of the beam of infrared radiation.
20. The system of claim 13 , wherein the infrared source is a broadband infrared source.
21. The system of claim 13 , wherein the infrared source is a tunable infrared source having a wavelength that is tunable between 2-25 microns.
22. The system of claim 13 , wherein the excitation radiation has a wavelength that is outside of a range of the infrared light source.
23. The system of claim 13 , wherein constructing the signal is performed by at least one of a lock-in amplifier, a notch filter, an RMS-to-DC converter, a Fourier transform, and a resonant amplifier.
24. The system of claim 13 , wherein the sample comprises at least one of a biological cell, a biological tissue, and a biological organism.