Fluorescence Imaging Flow Cytometry With Enhanced Image Resolution

Abstract

In one aspect, a system for performing flow cytometry is disclosed, which comprises a laser for generating laser radiation for illuminating a sample, at least one detector for detecting at least a portion of a radiation emanating from the sample in response to said illumination so as to generate a temporal signal corresponding to said detected radiation, and an analysis module for receiving said temporal signal and performing a statistical analysis of said signal based on a forward model to reconstruct an image of said sample.

1 - 49 . (canceled) 
     
     
         50 . A system comprising:
 a laser for generating laser radiation for illuminating a particle in a sample in a flow stream,   a light detection system comprising a photodetector configured to detect at least a portion of a radiation emanating from a particle in the sample in response to said illumination and to generate a temporal signal,   an analysis module operably connected to the detector, the analysis module comprising a processor having memory operably coupled to the processor wherein the memory comprises instructions stored thereon, which when executed by the processor cause the processor to:
 receive the temporal signal from the light detection system; and 
   perform an analysis of said temporal signal to reconstruct an image of the particle in said sample in the flow stream.   
     
     
         51 . The system according to  claim 50 , wherein the analysis of the temporal signal is a statistical analysis that is based on a forward model. 
     
     
         52 . The system according to  claim 51 , wherein the statistical analysis is used to refine the parameters of the model by minimizing the difference between the temporal signal generated by the photodetector and a corresponding temporal signal predicted by the forward model. 
     
     
         53 . The system according to  claim 50 , wherein the system comprises a light beam generator comprising the laser and an acousto-optic device configured to generate a frequency shifted beam of light. 
     
     
         54 . The system according to  claim 53 , wherein the acousto-optic device comprises an acousto-optic deflector (AOD). 
     
     
         55 . The system according to  claim 50 , wherein the image is any of a fluorescence, a darkfield, and a brightfield image. 
     
     
         56 . The system of  claim 50 , wherein the temporal signal is a fluorescence signal. 
     
     
         57 . The system of  claim 50 , wherein the temporal signal is a scattering signal. 
     
     
         58 . The system of  claim 50 , wherein the memory further comprises instructions to model the detected temporal signal as a plurality of temporal segments each having one sinusoidal and one cosinusoidal term. 
     
     
         59 . The system of  claim 50 , wherein the memory comprises instructions to perform an analysis that employs a least squares regression analysis so as to obtain values for parameters of said model of the temporal signal by minimizing a sum of squared residuals corresponding to differences between said modeled and the respective measured temporal segments. 
     
     
         60 . The system of  claim 50 , wherein the forward model comprises a non-linear model. 
     
     
         61 . The system of  claim 50 , wherein the memory comprises instructions to perform an analysis that comprises a gradient descent optimization method. 
     
     
         62 . The system of  claim 61 , wherein the memory comprises instructions to process the temporal signal to generate a fluorescence image and using said fluorescence image as a seed image for performing said statistical analysis. 
     
     
         63 . The system of  claim 61 , wherein the memory comprises instructions to perform an analysis that employs a least squares method to obtain said reconstructed image by minimizing a sum of squared residuals corresponding to a difference between said detected temporal signal and a respective temporal signal inferred from said forward model. 
     
     
         64 . The system of  claim 50 , wherein the memory comprises instructions to compute an initial estimated image via application of a Fast Fourier Transform (FFT) to the temporal signal. 
     
     
         65 . The system of  claim 50 , wherein the memory comprises instructions to perform an analysis that employs a priori information about the measured temporal signal in combination with Bayesian spectral estimation to reconstruct the image of the sample. 
     
     
         66 . The system of  claim 65 , wherein the a priori information indicates that the temporal signal is composed of a number of sinusoids of unknown frequencies and amplitudes. 
     
     
         67 . The system of  claim 65 , wherein the Bayesian spectral estimation provides estimates of said unknown frequencies and amplitudes of the sinusoids. 
     
     
         68 . The system of  claim 50 , wherein the memory comprises instructions to perform an analysis that employs:
 a particle swarm optimization method; or   a genetic algorithm.   
     
     
         69 . The system of  claim 50 , wherein the system further comprises a sorter for sorting the particle.