Video rate mid-infrared photothermal microscopy system using synchronized laser scanning
A mid-infrared photothermal microscopy system images a sample. A mid-infrared optical source generates a mid-infrared beam which is directed along a first optical path to reach the substrate on a first side and heat the sample. A probe light source generates a probe light which is directed along a second optical path to reach the substrate on a second side and illuminate the sample. A first laser scanner is positioned along the first optical path and configured to rotate to redirect light and scan the sample with the mid-infrared beam. A second laser scanner is positioned along the second optical path and configured to rotate to redirect light and scan the sample with the probe light. The laser scanners each include at least one mirror driven to rotate such that the mid-infrared beam and the probe light scan the sample synchronously.
1. A mid-infrared photothermal microscopy system for imaging a sample comprising:
a mid-infrared optical source configured to generate a mid-infrared beam, the mid-infrared beam being directed along a first optical path towards the sample to heat the sample;
a probe light source configured to generate a probe light, the probe light being directed along a second optical path towards the sample, the second optical path overlapping and counter-propagating with the first optical path at the sample;
a first laser scanner comprising at least one movable mirror, positioned along the first optical path and configured to rotate to redirect light and scan the sample with the mid-infrared beam;
a second laser scanner comprising at least one movable mirror, positioned along the second optical path and configured to rotate to redirect light and scan the sample with the probe light,
a reflective objective lens arranged along the first optical path between the mid-infrared optical source and the sample; and
a second objective lens arranged along the second optical path between the probe light source and the sample,
wherein the laser scanners are driven to rotate such that the mid-infrared beam and the probe light scan the sample synchronously.
2. The system of claim 1 , further comprising a photodiode configured to detect probe light from the sample to generate a reconstructed image of the sample.
3. The system of claim 2 , wherein the mid-infrared beam is a pulsed beam and wherein the MIP system further comprises single pulse photothermal detection.
4. The system of claim 1 , wherein:
the first laser scanner comprises a first pair of scanning mirrors, including a first mirror and a second mirror; and
the second laser scanner comprises a second pair of scanning mirrors, including a third mirror and a fourth mirror.
5. The system of claim 4 , wherein the first pair of scanning mirrors have orthogonal scanning axes; and
the second pair of scanning mirrors have orthogonal scanning axes.
6. The system of claim 1 , further comprising a digital-to-analog device configured to generate four control signals to drive the laser scanners for synchronous scanning of the mid-infrared beam and probe lights.
7. The system of claim 6 , wherein at least one of the four control signals is adjusted by an angle scaling factor based on the focal lengths of probe light and mid-infrared objectives and the beam expansion ratio of relay systems, the angle scaling factor determining relative angular motion for the laser scanners for the probe light and the mid-infrared beam.
8. The system of claim 1 , wherein a relative scaling of motion for the mid-infrared and probe light laser scanning mechanisms is configured such that the probe light and mid-infrared beam are focused to overlapping locations on the sample during scanning.
9. The system of claim 1 , further comprising:
a first photodiode positioned along the first optical path and configured to detect the probe light passing through the sample; and
a second photodiode positioned along the second optical path and configured to detect the probe light returning after reflecting off the sample.
10. The system of claim 1 , wherein the mid-infrared beam is pulsed with a repetition rate between 500 kHz to 1 MHz and a duty cycle of less than 30%.
11. The system of claim 1 , wherein the pairs of laser scanners are configured to cause the sample to be scanned at a frequency of substantially 3 kHz.
12. The system of claim 1 , further comprising a scan lens positioned on the second optical path between the second pair of laser scanners and the substrate, wherein the probe light is conjugated to a back pupil of the second objective lens with the scan lens.
13. The system of claim 12 , further comprising a tube lens positioned on the second optical path between the second objective lens and the scan lens, wherein the tube lens introduces a beam expansion to substantially fill the back pupil of the objective lens.
14. The system of claim 1 , wherein a focus of the mid-infrared beam is aligned to overlap with a focus of the probe light.
15. The system of claim 1 , wherein at least one of the laser scanners comprises at least one galvo mirror.
16. The system of claim 1 , wherein the probe light and mid-infrared beam are scanned across a shared focal spot of the sample as each of the laser scanners rotates in both a forward direction and a backward direction, the backward direction being opposite the forward direction.
17. The system of claim 16 , wherein rotational movement of the laser scanners follows a sinusoidal pattern.
18. The system of claim 1 , further comprising a laser scanning position sensor configured to measure a position of at least one of the laser scanners during scanning, wherein a reconstructed image of the sample is adjusted based on feedback from the galvo position sensor.
19. The system of claim 1 , wherein the laser scanners provide an effective scan area of the mid-IR and probe lights of at least 100 micrometers on a side.
20. The system of claim 1 , wherein the laser scanners provide an effective scan area of the mid-IR and probe lights of at least 200 micrometers on a side.
21. A method of operating a photothermal infrared microscope to image a sample, the method comprising:
illuminating the sample with a beam of mid-infrared light to heat the sample by directing the beam of infrared light along a first optical path towards the sample;
illuminating the sample with a beam of probe light by directing the probe light along a second optical path towards the sample, the second optical path overlapping and counter-propagating with the first optical path at the sample;
providing a first laser scanner comprising at least one movable mirror, positioned along the first optical path and configured to rotate to redirect light and scan the sample with the mid-infrared beam;
providing a second laser scanner comprising at least one movable mirror, positioned along the second optical path and configured to rotate to redirect light and scan the sample with the probe light;
providing a reflective objective lens arranged along the first optical path between the mid-infrared optical source and the sample; and
providing a second objective lens arranged along the second optical path between the probe light source and the sample;
wherein the laser scanners are driven to rotate such that the mid-infrared beam and the probe light scan the sample synchronously.
22. The method of claim 21 , further comprising collecting an image of mid-infrared absorption by the sample covering an area at least 10 microns on a side in a time of 0.1 seconds or less.
23. The method of claim 21 , further comprising measuring cellular dynamics within a biological cell.
24. The method of claim 21 , further comprising:
collecting probe light from the sample; and
detecting a change in probe light collected from the sample at the plurality of locations on the sample in response to radiation from the mid-infrared light absorbed by the sample.
25. The method of claim 24 , further comprising extracting a signal from the detected change in collected probe light to produce an image that is indicative of infrared absorption by the sample.
26. The method of claim 25 , further comprising collecting a plurality of images at a plurality of mid-infrared wavelengths.
27. The method of claim 25 , further comprising measuring a plurality of images at different times to record dynamic changes in the sample.
28. The method of claim 25 , wherein the image indicative of infrared absorption by the sample has a signal to noise ratio of greater than 50.