Parallel flow cytometer using radiofrequency multiplexing
An imaging flow cytometry apparatus and method which allows registering multiple locations across a cell, and/or across multiple flow channels, in parallel using radio-frequency-tagged emission (FIRE) coupled with a parallel optical detection scheme toward increasing analysis throughput. An optical source is modulated by multiple RF frequencies to produce an optical interrogation beam having a spatially distributed beat frequency. This beam is directed to one or more focused streams of cells whose responsive fluorescence, in different frequencies, is registered in parallel by an optical detector.
1. An apparatus comprising:
a light beam generator component configured to generate at least a first beam of frequency shifted light and a second beam of frequency shifted light, wherein the first beam of frequency shifted light comprises a modulation frequency that is different from the second beam of frequency shifted light;
an optical apparatus configured to simultaneously direct the first beam of frequency shifted light onto a first flow channel and the second beam of frequency shifted light onto a second flow channel; and
a photodetector.
2. The apparatus according to claim 1 , wherein the light beam generator component comprises:
a first radio-frequency source having a first radio-frequency output;
a second radio-frequency source having a second radio-frequency output; and
an acousto-optical combiner configured to combine the first radio-frequency output and the second radio-frequency output to produce an optical interrogation beam comprising the first beam of frequency shifted light and the second beam of frequency shifted light.
3. The apparatus according to claim 2 , wherein first beam of frequency shifted light is spatially distributed from the second beam of frequency-shifted light.
4. The apparatus according to claim 2 , wherein the photodetector is configured to simultaneously detect fluorescence from particles at a first modulation frequency moving through the first flow channel and fluorescence from particles at a second modulation frequency moving through the second flow channel.
5. The apparatus according to claim 4 , wherein fluorescence detected at the first modulation frequency and fluorescence detected at the second modulation frequency are registered in parallel.
6. The apparatus according to claim 4 , wherein the photodetector is configured to simultaneously detect fluorescence from particles moving at a rate of 1 m/s through the first flow channel and the second flow channel.
7. The apparatus according to claim 2 , wherein the acousto-optical combiner is configured to independently control the intensity of the first beam of frequency shifted light and the intensity of the second beam of frequency shifted light.
8. The apparatus according to claim 7 , wherein the first flow channel and the second flow channel have substantially the same optical sensitivity.
9. The apparatus according to claim 1 , wherein the light beam generator comprises one or more of an acousto-optic deflector (AOD) and an acousto-optic frequency shifter (AOFS).
10. The apparatus according to claim 9 , further comprising a radio-frequency comb generator configured to produce the first beam of frequency shifted light and the second beam of frequency shifted light, wherein the first beam of frequency shifted light and the second beam of frequency shifted light are amplitude modulated and spatially disparate.
11. The apparatus according to claim 10 , wherein the first beam of frequency shifted light and the second beam of frequency shifted light have a spatial width sufficient to span the width of the first flow channel and the width of the second flow channel.
12. The apparatus according to claim 1 , wherein the light beam generator comprises a laser.
13. The apparatus according to claim 12 , wherein the laser is a continuous wave laser.
14. The apparatus according to claim 1 , further comprising a microfluidic device comprising a plurality of parallel flow channels,
wherein the light beam generator component is configured to generate an interrogation beam comprising a plurality of spatially distributed beams of frequency shifted light; and
wherein the optical apparatus is configured to direct each of the plurality of beams of frequency shifted light onto the plurality of parallel flow channels.
15. The apparatus according to claim 1 , wherein the photodetector is a single photomultiplier tube (PMT).
16. A method comprising:
irradiating with a first beam of frequency shifted light a first sample composition comprising particles moving through a first flow channel and irradiating with a second beam of frequency shifted light a second sample composition comprising particles moving through a second flow channel, wherein the first beam of frequency shifted light comprises a modulation frequency that is different from the second beam of frequency shifted light; and
detecting light from particles moving through the first flow channel and from particles moving through the second flow channel.
17. The method according to claim 16 , wherein fluorescence light is detected simultaneously from particles moving through the first flow channel and from particles moving through the second flow channel.
18. The method according to claim 16 , wherein fluorescence detected at the first modulation frequency and fluorescence detected at the second modulation frequency are registered in parallel.