Flow Cytometer With Optical Equalization

Abstract

Aspects of the present disclosure include methods for producing an output laser beam having two or more angularly deflected laser beams (e.g., for irradiating a sample in a flow stream) with a predetermined intensity profile. Systems for practicing the subject methods having a laser, an acousto-optic device, a radiofrequency generator and a controller for adjusting the amplitude of the radiofrequency drive signals to produce an output laser beam of angularly deflected laser beams with a predetermined intensity profile are also described.

1 .- 108 . (canceled) 
     
     
         109 . A method comprising:
 generating with a light beam generator a first plurality of angularly deflected laser beams and a second plurality of angularly deflected laser beams; and   optically combining the first plurality of angularly deflected laser beams with the second plurality of angularly deflected laser beams in a manner such that the first plurality of angularly deflected laser beams at least partially overlap with the second plurality of angularly deflected laser beams.   
     
     
         110 . The method according to  claim 109 , wherein the first plurality of angularly deflected laser beams and the second plurality of angularly deflected laser beams are generated by splitting a laser beam outputted from an acousto-optic device irradiated by a laser. 
     
     
         111 . The method according to  claim 110 , wherein the first plurality of angularly deflected laser beams are identical to the second plurality of angularly deflected laser beams. 
     
     
         112 . The method according to  claim 109 , wherein the angularly deflected laser beams are aligned along a horizontal axis. 
     
     
         113 . The method according to  claim 109 , wherein the method comprises inverting the second plurality of angularly deflected laser beams and optically combining the inverted second plurality of angularly deflected laser beams with the first plurality of angularly deflected laser beams. 
     
     
         114 . The method according to  claim 109 , wherein the first plurality of angularly deflected laser beams and second plurality of angularly deflected laser beams have a predetermined intensity profile along a horizontal axis. 
     
     
         115 . The method according to  claim 114 , wherein the intensity profile comprises increasing intensity from the center to the edges of the angularly deflected laser beams along the horizontal axis. 
     
     
         116 . The method according to  claim 115 , wherein the intensity at the center of the angularly deflected laser beams is from 0.1% to 99% of the intensity at the edge of the angularly deflected laser beams along the horizontal axis. 
     
     
         117 . The method according to  claim 113 , wherein the intensity profile comprises an increasing intensity from the edges to the center of the angularly deflected laser beams along the horizontal axis. 
     
     
         118 . The method according to  claim 109 , wherein the method comprises:
 applying a plurality of radiofrequency drive signals to an acousto-optic device; and   irradiating the acousto-optic device with the laser to generate an output laser beam comprising a plurality of angularly deflected laser beams.   
     
     
         119 . The method according to  claim 118 , wherein the amplitude of the first plurality of angularly deflected laser beams and the second plurality of angularly deflected laser beams is based on the amplitude of the radiofrequency drive signals applied to the acousto-optic device. 
     
     
         120 . The method according to  claim 109 , wherein the method further comprises:
 irradiating a sample propagated in a flow stream with the combined first plurality of angularly deflected laser beams and second plurality of angularly deflected laser beams; and   detecting light from the irradiated sample in the flow stream.   
     
     
         121 . The method according to  claim 120 , wherein the method comprises measuring the detected light at one or more wavelengths. 
     
     
         122 . The method according to  claim 120 , wherein the method comprises monitoring the detected light from the irradiated sample in the flow stream to detect spatial drift of the combined first plurality of angularly deflected laser beams and second plurality of angularly deflected laser beams. 
     
     
         123 . The method according to  claim 122 , further comprising adjusting the spatial position of the combined first plurality of angularly deflected laser beams and second plurality of angularly deflected laser beams on the flow stream when spatial drift is detected. 
     
     
         124 . The method according to  claim 123 , wherein adjusting the spatial position comprises adjusting the frequency of the applied radiofrequency drive signals. 
     
     
         125 . The method according to  claim 118 , wherein the method further comprises:
 generating a detection signal in response to light from the irradiated flow stream; and   frequency demultiplexing the detection signal to determine one or more beat frequencies.   
     
     
         126 . The method according to  claim 125 , wherein each of the beat frequencies corresponds to a frequency difference between a pair of overlapping beams from the first plurality of angularly deflected laser beams with the second plurality of angularly deflected laser beams. 
     
     
         127 . The method according to  claim 126 , wherein the method further comprises normalizing intensity of the generated data signal based on the spatial location of each overlapping beam of the first plurality of angularly deflected laser beams and second plurality of angularly deflected laser beams and a frequency content of a forward scattered light data signal.