Method for analyzing and selecting a specific droplet among a plurality of droplets and associated apparatus

Abstract

The present invention relates to a method for analyzing and selecting a specific droplet among a plurality of droplets (4), comprising the following steps: —providing a plurality of droplets (4), —for a droplet (4) among the plurality of droplets, measuring at least two optical signals, each optical signal being representative of a light intensity spatial distribution in the droplet for an associated wavelength channel, —calculating a plurality of parameters from the optical signals, —determining a sorting class for a droplet according to calculated parameters, —sorting said droplet according to its sorting class, wherein the plurality of parameters comprises the coordinates of a maximum for each optical signal and a co-localization parameter and the at least two calculated parameters used for the determining step comprises the co-localization parameter.

1 . A method for analyzing and selecting a specific droplet among a plurality of droplets ( 4 ), comprising the following steps:
 providing a plurality of droplets ( 4 ),   for a droplet ( 4 ) among the plurality of droplets, measuring at least two optical signals, each optical signal being representative of a light intensity spatial distribution in the droplet ( 4 ) for an associated wavelength channel,   calculating a plurality of parameters from the at least two optical signals,   determining a sorting class for a droplet according to at least two calculated parameters,   sorting said droplet according to its sorting class,   
       wherein the plurality of parameters comprises the coordinates of a maximum for each optical signal and a co-localization parameter (Δ) and the at least two calculated parameters used for the determining step comprises the co-localization parameter (Δ). 
     
     
         2 . A method according to  claim 1 , wherein a co-localization parameter (Δ) is calculated by comparing the position corresponding to the maximum intensity of a first optical signal among the at least two optical signals to the position corresponding to the maximum intensity of a second optical signal among the at least two optical signals. 
     
     
         3 . A method according to any of the  claims 1  and  2 , wherein the co-localization parameter (Δ) is selected by having a confidence interval ranging from 90% to 100%. 
     
     
         4 . A method according to any of the  claims 1  to  3 , wherein the plurality of parameters comprises at least one of the following parameters:
 a droplet ( 4 ) width, 
 an integration of an optical signal, 
 a ratio between a maximum value of an optical signal and an integration value of said optical signal, 
 the coordinates of a local maximum for an optical signal, 
 the number of local maxima in a droplet for an optical signal, 
 the calculation of the derivative of an optical signal and the calculation of the second derivative for an optical signal. 
 
     
     
         5 . A method according to any of the preceding claims, wherein a first optical signal among the at least two optical signals comprises a plurality of local maxima, the plurality of parameter comprises a multipeak co-localization parameter calculated between the first optical signal and a second optical signal comprising a local maximum, the multipeak co-localization parameters being calculated with the following steps:
 for each local maximum of the first optical signal, calculating an intermediate co-localization parameter, by comparing the position of the local maximum of the second signal to the position of said local maximum of the first optical signal,   comparing the intermediate co-localization parameters, the multipeak co-localization parameter being the lowest intermediate co-localization parameter.   
     
     
         6 . A method according to any of the preceding claims, wherein a co-localization parameter in a droplet ( 6 ) is normalized by the droplet width. 
     
     
         7 . A method according to any of the preceding claims, wherein the step of measuring is performed for at least two droplets ( 4 ) of the plurality of droplets ( 4 ) and the plurality of parameters comprises the spacing between the two droplets ( 4 ). 
     
     
         8 . A method according to any of the preceding claims wherein during the determining step, at least a calculated parameter is compared to predetermined threshold values. 
     
     
         9 . A method according to any of the preceding claims, wherein, during the measuring step, at least three optical signals are measured, and wherein a plurality of co-localization parameters are calculated by comparing the position of the maximum of the optical signals two by two. 
     
     
         10 . A method according to any of the preceding claims further comprising the following step:
 providing an apparatus ( 1 ) comprising a channel ( 30 ) adapted for a flow of droplets ( 4 ), the apparatus ( 1 ) comprising a detection area ( 34 ), and a sorting area ( 32 ),   the plurality of droplets ( 4 ) circulating in the channel ( 30 ),   carrying out a measurement for a droplet ( 4 ) flowing in the detection area ( 34 ).   
     
     
         11 . A method according to any of the preceding claims comprising a step of capturing a picture of the droplet ( 4 ) during the measuring step. 
     
     
         12 . A method according to any of the preceding claims wherein at least a droplet ( 6 ) of the plurality of droplets ( 4 ) comprises a first element ( 26 ), the first element ( 26 ) being fluorescent in a wavelength channel associated to a first optical signal among the at least two optical signals, and wherein at least a droplet ( 4 ) of the plurality of droplets ( 4 ) comprises a second element ( 28 ), the second element ( 28 ) being fluorescent in a second wavelength channel associated to a second optical signal among the at least two optical signals. 
     
     
         13 . A method according to  claim 12 , wherein the first and second element ( 26 ,  28 ) are chosen in the group of elements consisting of a cell, a fluorescently labelled protein, a cell labelling reagent, a fluorescently labeled antigen, a fluorescently labelled antibody, a particle coated with a biological entity, a nucleic acid, a peptide and a chemical drug. 
     
     
         14 . An apparatus ( 1 ) for analyzing and selecting a specific droplet ( 4 ) among a plurality of droplets ( 4 ) comprising:
 a detection assembly ( 12 ) adapted to measure, for a droplet ( 4 ), at least two optical signals, each optical signal being representative of a light intensity spatial distribution in the droplet ( 4 ) for an associated wavelength channel,   a calculator ( 14 ) for calculating a plurality of parameters from the at least two optical signals,   a selecting unit ( 16 ) for determining a sorting class for the droplet ( 4 ) according to at least two calculated parameters,   a sorting unit ( 18 ) for sorting the droplet ( 4 ) according to its sorting class, wherein the plurality of parameter comprises the coordinate of the maximum for each optical signal and a co-localization parameter and the at least two calculated parameters comprises the co-localization parameter.   
     
     
         15 . An apparatus ( 1 ) according to  claim 14 , wherein the detection assembly comprises a light source ( 36 ) and at least a visible light sensitive detector ( 38 ).