Wide area optical photothermal infrared spectroscopy
Apparatuses and methods for microscopic analysis of a sample by simultaneously characterizing infrared absorption characteristics of a plurality of spatially resolved locations are described herein. These apparatuses and methods improve sampling times while collecting microscopic data regarding composition of a sample across a wide field.
1. An apparatus for microscopic analysis of a sample by simultaneously characterizing infrared absorption characteristics of a plurality of spatially resolved locations corresponding to a wide area of the sample, the apparatus comprising:
a source of infrared radiation configured to illuminate the wide area of the sample with a beam of infrared radiation;
a source of probe radiation configured to illuminate the wide area of the sample with a beam of probe radiation;
a collector configured to collect as collected probe radiation at least a portion of probe radiation from each of the plurality of spatially resolved locations on the sample;
at least one camera configured to detect at least a portion of the collected probe radiation to generate signals indicative of infrared absorption corresponding to each one of the plurality of spatially resolved locations,
wherein the signals indicative of infrared absorption have a spatial resolution of less than 1 micrometer.
2. The apparatus of claim 1 wherein the source of infrared radiation is tunable to produce an infrared beam with variable wavelength and wherein the signals indicative of infrared absorption of the sample are detected at a plurality of infrared wavelengths.
3. The apparatus of claim 2 wherein the signals indicative of infrared absorption at a plurality of infrared wavelengths comprise infrared absorption spectra.
4. The apparatus of claim 1 further comprising an image co-adder wherein the image co-adder sums multiple camera frames to construct a co-added image with a dynamic range of at least 10 4 .
5. The apparatus of claim 1 where the co-added image has a dynamic range of at least 10 5 .
6. The apparatus of claim 1 where the co-added image has a dynamic range of at least 10 6 .
7. The apparatus of claim 1 wherein the plurality of sample locations measured simultaneously comprise an area of at least 100 μm in diameter.
8. The apparatus of claim 1 wherein the signals indicative of infrared absorption of the plurality of sample locations achieve a spatial resolution of less than 0.5 μm.
9. The apparatus of claim 1 further comprising a gate function that limits a duration of time over which the camera detects collected probe radiation from the plurality of sample locations.
10. The apparatus of claim 9 wherein the gate function limits at least one of: (a) an exposure time of the camera; and (b) a pulse duration length of the probe beam.
11. The apparatus of claim 1 wherein signals indicative of infrared absorption of the plurality of sample locations are generated by comparing a first set of camera frames collected with the infrared source irradiating the sample and a second set of camera frames collected with the infrared source not irradiating the sample.
12. The apparatus of claim 1 wherein the collector comprises an objective with a numerical aperture (NA) of at least 0.4.
13. The apparatus of claim 1 wherein the collector comprises an objective with a numerical aperture (NA) of at least 0.6.
14. The apparatus of claim 1 wherein the source of probe radiation is an incoherent light source.
15. The apparatus of claim 1 wherein the source of probe radiation comprises at least one light emitting diode.
16. The apparatus of claim 1 , wherein generating signals indicative of infrared absorption corresponding to each one of the plurality of spatially resolved locations is based on measuring fluctuations in phase of the collected probe light caused by infrared absorption at the sample.
17. A method of operating a system for simultaneously characterizing infrared absorption characteristics of a plurality of locations on a sample, the method comprising the steps of:
illuminating the plurality of locations of the sample with a source of infrared radiation to create an infrared illuminated spot on the sample;
illuminating the plurality of locations of the sample with a source of probe radiation;
collecting probe radiation from the plurality of sample locations;
detecting collected probe radiation with at least one camera; and
analyzing camera detected probe radiation to generate signals indicative of infrared absorption of the plurality of locations on the sample, wherein the signals indicative of infrared absorption of the plurality of sample locations achieve a spatial resolution of less than 1 μm.
18. The method of claim 17 further comprising tuning the source of infrared radiation to produce an infrared beam having a variable wavelength and wherein the signals indicative of infrared absorption of the sample are detected at a plurality of infrared wavelengths.
19. The method of claim 17 wherein the signals indicative of infrared absorption comprise infrared absorption spectra.
20. The method of claim 19 wherein the infrared absorption spectra are measured at a rate exceeding 20 spectra per second.
21. The method of claim 19 wherein the infrared absorption spectra are measured at a rate exceeding 50 spectra per second.
22. The method of claim 19 wherein the infrared absorption spectra are measured at a rate exceeding 90 spectra per second.
23. The method of claim 17 , wherein the system further comprises an image co-adder and the method further comprises, using the co-adder, combining multiple camera frames to construct a co-added image with a dynamic range of at least 10 4 .
24. The method of claim 23 wherein the co-added image has a dynamic range of at least 10 5 .
25. The method of claim 23 wherein the co-added image has a dynamic range of at least 10 6 .
26. The method of claim 17 further comprising simultaneously measuring an area of at least 100 μm in diameter.
27. The method of claim 17 wherein the signals indicative of infrared absorption of the plurality of sample locations achieve a spatial resolution of less than 0.5 μm.
28. The method of claim 17 further comprising applying a gate function to limit a duration of time over which the camera detects collected probe radiation from the plurality of sample locations.
29. The method of claim 28 wherein the gate function limits at least one of: (a) an exposure time of the camera; and (b) a pulse duration length of the probe beam.
30. The method of claim 29 wherein the signals indicative of infrared absorption of the plurality of sample locations achieve a spatial resolution of less than 0.5 μm.
31. The method of claim 17 wherein signals indicative of infrared absorption of the plurality of sample locations are generated by comparing a first set of camera frames collected with the infrared source irradiating the sample and a second set of camera frames collected with the infrared source not irradiating the sample.
32. The method of claim 17 , wherein generating signals indicative of infrared absorption corresponding to each one of the plurality of spatially resolved locations is based on measuring fluctuations in phase of the collected probe light caused by infrared absorption at the sample.