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:
an infrared light source configured to illuminate the wide area of the sample with a beam of infrared radiation;
a reflective objective arranged to receive the beam of infrared radiation from the infrared light source and illuminate the wide area of the sample at a first side thereof;
a probe light source configured to generate a beam of probe radiation;
a refractive objective arranged to receive the beam of probe radiation and illuminate the wide area of the sample at a second side thereof
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.
2. The apparatus of claim 1 wherein the infrared radiation source is tunable to produce the infrared beam with a 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 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.
9. The apparatus of claim 8 wherein the gate function limits at least one of: (a) an exposure time of the camera; and (b) a pulse duration length of the beam of probe radiation.
10. 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 light source irradiating the sample and a second set of camera frames collected with the infrared light source not irradiating the sample.
11. The apparatus of claim 1 wherein the collector comprises an objective with a numerical aperture (NA) of at least 0.4.
12. The apparatus of claim 1 wherein the collector comprises an objective with a numerical aperture (NA) of at least 0.6.
13. The apparatus of claim 1 wherein the probe light source is an incoherent light source.
14. The apparatus of claim 1 wherein the probe light source comprises at least one light emitting diode.
15. 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.
16. A method of operating a system for simultaneously characterizing infrared absorption characteristics of plurality of locations on a sample, the method comprising the steps of:
illuminating the plurality of locations of the sample with a reflective objective and an infrared light source that are arranged to illuminate a first side of the sample;
illuminating the plurality of locations of the sample with a refractive objective and a probe light source that are arranged to illuminate a second side of the sample;
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.
17. The method of claim 16 further comprising tuning the infrared light source to produce the 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.
18. The method of claim 16 wherein the signals indicative of infrared absorption comprise infrared absorption spectra.
19. The method of claim 18 wherein the infrared absorption spectra are measured at a rate exceeding 20 spectra per second.
20. The method of claim 18 wherein the infrared absorption spectra are measured at a rate exceeding 50 spectra per second.
21. The method of claim 18 wherein the infrared absorption spectra are measured at a rate exceeding 90 spectra per second.
22. The method of claim 16 , 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 .
23. The method of claim 22 wherein the co-added image has a dynamic range of at least 10 5 .
24. The method of claim 22 wherein the co-added image has a dynamic range of at least 10 6 .
25. The method of claim 16 further comprising simultaneously measuring an area of at least 100 μm in diameter.
26. The method of claim 16 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.
27. The method of claim 26 wherein the gate function limits at least one of: (a) an exposure time of the camera; and (b) a pulse duration length of the beam of probe radiation.
28. The method of claim 16 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 light source irradiating the sample and a second set of camera frames collected with the infrared light source not irradiating the sample.
29. The method of claim 16 , 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.