Multi-modal fluorescence imaging flow cytometry system
In one aspect, the present teachings provide a system for performing cytometry that can be operated in three operational modes. In one operational mode, a fluorescence image of a sample is obtained by exciting one or more fluorophore(s) present in the sample by an excitation beam formed as a superposition of a top-hat-shaped beam with a plurality of beams that are radiofrequency shifted relative to one another. In another operational mode, a sample can be illuminated successively over a time interval by a laser beam at a plurality of excitation frequencies in a scanning fashion. The fluorescence emission from the sample can be detected and analyzed, e.g., to generate a fluorescence image of the sample. In yet another operational mode, the system can be operated to illuminate a plurality of locations of a sample concurrently by a single excitation frequency, which can be generated, e.g., by shifting the central frequency of a laser beam by a radiofrequency. For example, a horizontal extent of the sample can be illuminated by a laser beam at a single excitation frequency. The detected fluorescence radiation can be used to analyze the fluorescence content of the sample, e.g., a cell/particle.
1. A method comprising:
generating an angularly deflected laser beam and a plurality of radiofrequency-shifted laser beams with an acousto-optic device;
propagating the angularly deflected laser beam along a first optical path;
propagating the plurality of radiofrequency-shifted laser beams along a second optical path;
irradiating a sample with the angularly deflected laser beam and the plurality of radiofrequency-shifted laser beams, wherein the angularly deflected laser beam overlaps each of the plurality of radiofrequency-shifted laser beams on the irradiated sample; and
detecting light from the sample.
2. The method according to claim 1 , wherein acousto-optic device is an acousto-optic deflector.
3. The method according to claim 1 , wherein the angularly deflected laser beam comprises a local oscillator (LO) beam.
4. The method according to claim 3 , wherein the method comprises propagating the local oscillator beam through a top-hat beam shaper to produce a local oscillator beam with a substantially constant intensity profile along a horizontal axis.
5. The method according to claim 4 , wherein the local oscillator beam has a Gaussian intensity profile along a vertical axis.
6. The method according to claim 1 , wherein light emitted from the sample where each radiofrequency-shifted laser beam overlaps with the angularly deflected beam exhibits a beat frequency that comprises a frequency difference between the angularly deflected laser beam and each radiofrequency comb beam.
7. The method according to claim 1 , further comprising modulating the intensity of each of the radiofrequency comb beams.
8. The method according to claim 7 , wherein modulating the intensity of the radiofrequency comb beams comprises adjusting the intensity of each radiofrequency comb beam to be equal to the intensity of the angularly deflected laser beam.
9. The method according to claim 1 , wherein the method further comprises generating a data signal in response to the detected light.
10. The method according to claim 9 , wherein the method comprises generating a plurality of time-domain fluorescence emission signals.
11. The method according to claim 10 , further comprising generating an image of the sample based on the plurality of time-domain fluorescence emission signals.
12. The method according to claim 11 , wherein generating the image comprises frequency de-multiplexing each fluorescence emission signal to determine beat frequencies where each radiofrequency-shifted laser beam overlaps with the angularly deflected beam.
13. The method according to claim 12 , wherein frequency de-multiplexing comprises obtaining a Fourier Transform of the fluorescence signal to determine the beat frequencies.
14. The method according to claim 13 , wherein generating the image comprises computing the amplitude of each beat frequency to provide a pixel value corresponding to a location of the image.
15. The method according to claim 14 , wherein generating the image comprises generating a darkfield image of the sample and generating a brightfield image of the sample.
16. The method according to claim 1 , wherein the sample is present in a flow stream.
17. The method according to claim 16 , wherein the flow stream has a flow rate of particles of 1 m/s or more.
18. The method according to claim 1 , wherein the sample comprises one or more fluorophores.