Temporal Multiplexed Excitation for Miniaturized, Planar Fluorescence Activated Cell Sorting

Abstract

A system for fluorescence activated cell sorting includes at least two excitation lasers and an objective that directs light from the at least two excitation lasers to a common point in an interrogation region of a fluidic channel. The fluidic channel directs a flow of a plurality of fluorescently labeled particles through the interrogation region. At least one modulator temporally multiplexes light from the at least two excitation lasers such that pulses of light from different lasers intersect the common point at different times. The system further includes at least one detector and at least one optical element that directs light emitted from the particles and transmitted through the objective to the at least one detector. The system may further include optics for generating and detecting side and forward scattered light. Methods for operating example systems to collect fluorescent, side scattered and forward scattered light are also described herein.

1 . A system comprising:
 at least two excitation lasers;   an objective that directs light from the at least two excitation lasers to a common point in an interrogation region of a fluidic channel, wherein the fluidic channel directs a flow of a plurality of fluorescently labeled particles through the interrogation region;   at least one modulator that temporally multiplexes light from the at least two excitation lasers such that pulses of light from different lasers intersect the common point in the interrogation region of the fluidic channel at different times;   at least one detector; and   at least one optical element optically coupled to the objective and the at least one detector to direct light emitted from the plurality of fluorescently labeled particles and transmitted through the objective to the at least one detector.   
     
     
         2 . The system of  claim 1 , wherein the fluidic channel is defined in a planar microfluidic chip. 
     
     
         3 . The system of  claim 1 , wherein the at least one modulator sequentially pulses the at least two excitation lasers. 
     
     
         4 . The system of  claim 1 , wherein the at least one detector comprises at least one single pixel detector. 
     
     
         5 . The system of  claim 1 , wherein the at least one detector comprises a single pixel detector for each wavelength of light emitted by the plurality of fluorescently-labeled particles. 
     
     
         6 . The system of  claim 1 , wherein the at least one optical element transmits incident light from each of the at least two excitation lasers and reflects light emitted by the plurality of fluorescently-labeled particles. 
     
     
         7 . The system of  claim 6 , wherein the at least one optical element comprises a dichroic having narrow transmission peaks for each wavelength of light incident by the at least two excitation lasers. 
     
     
         8 . The system of  claim 6 , wherein the at least one optical element comprises a dot optic. 
     
     
         9 . The system of  claim 1 , wherein the at least one optical element reflects incident light from each of the at least two excitation lasers and transmits light emitted by the plurality of fluorescently-labeled particles. 
     
     
         10 . The system of  claim 9 , wherein the at least one optical element comprises a dichroic having narrow transmission peaks for each wavelength of light emitted by the plurality of fluorescently-labeled particles. 
     
     
         11 . The system of  claim 9 , wherein the at least one optical element comprises a dot mirror. 
     
     
         12 . The system of  claim 1 , wherein the at least two excitation lasers comprises at least three excitation lasers. 
     
     
         13 . The system of  claim 1 , further comprising a detector for detecting forward scattered light transmitted through the fluidic channel. 
     
     
         14 . The system of  claim 1  further comprising at least one detector for detecting side scattered light reflected from the fluidic channel. 
     
     
         15 . The system of  claim 14 , further comprising at least one optical element for directing a portion of the light from one of the at least two lasers to the fluidic channel at a position upstream of the interrogation region with respect to the direction of flow of the plurality of fluorescently labeled particles in the fluidic channel. 
     
     
         16 . A method comprising:
 moving a plurality of fluorescently labeled particles through a fluidic channel comprising an interrogation region, wherein the plurality of fluorescently labeled particles move through the interrogation region at a flow speed;   directing light from at least two excitation lasers through an objective to a common point in the interrogation region of the fluidic channel;   temporally multiplexing light from the at least two excitation lasers such that pulses of light from different lasers intersect the common point in the interrogation region of the fluidic channel at different times;   receiving, by at least one detector, light emitted from each of the plurality of fluorescently labeled particles and transmitted through the objective; and   generating, by the at least one detector, a fluorescence signal indicative of intensity of light emitted from each of the plurality of fluorescently labeled particles as the particles move through the interrogation region.   
     
     
         17 . The method according to  claim 16 , further comprising:
 directing a portion of the light from one of the at least two lasers to the fluidic channel at a position upstream of the interrogation region with respect to the direction of flow in the fluidic channel; and   receiving, by at least one side scatter detector, side scattered light from each of the plurality of fluorescently labeled particles;   generating a side scattered signal by the at least one side scatter detector;   receiving, by at least one forward scatter detector, forward scattered light from each of the plurality of fluorescently labeled particles; and   generating a forward scatter signal by the at least one forward-scatter detector.   
     
     
         18 . The method of  claim 17 , wherein the side scattered light is emitted from within the fluidic channel. 
     
     
         19 . The method of  claim 17 , wherein the forward scattered light is transmitted through the fluidic channel. 
     
     
         20 . The method of  claim 17 , further comprising:
 synchronizing the fluorescence signal, forward scatter signal and side scatter signal attributed to a single particle of the plurality of particles based, at least in part, on the flow speed.