Systems and methods for generation of emulsions with suitable clarity with applications of use

Abstract

The inventions cover systems and methods for generation of emulsions having suitable clarity without requiring refractive index matching between emulsion components. Systems can include: a substrate including a set of openings; a reservoir facing the substrate at a first side and containing a sample fluid configured for droplet formation upon interacting with the set of openings of the substrate; and a collecting container facing the substrate at a second side and containing a set of fluid layers configured with a density gradient and suitable immiscibility characteristics. One or more components of the system(s) can support methods for emulsion generation, in relation to enabling interactions between multiple continuous phases and a dispersed droplet phase to generate clear emulsions. Applications of the inventions(s) can include performance of droplet-based digital PCR in an improved manner (e.g., without requiring implementation of correction factors based upon Poisson statistics).

What is claimed is: 
     
         1 - 21 . (canceled) 
     
     
         22 . A method comprising:
 performing a digital analysis for an antimicrobial susceptibility test of a sample distributed across a set of partitions,   wherein the sample comprises bacteria and an antibiotic, and   wherein performing the digital analysis comprises imaging a set of cross-sections of a closed container containing the set of partitions, and wherein the digital analysis for a cell replication cycle stage of the antimicrobial susceptibility test is performed within a duration of three minutes.   
     
     
         23 . The method of  claim 22 , wherein the sample further comprises a fluorescent growth indicator. 
     
     
         24 . The method of  claim 23 , wherein the fluorescent growth indicator comprises resazurin. 
     
     
         25 . The method of  claim 22 , wherein the set of partitions comprises a set of droplets of an emulsion generated using the sample, wherein clarity of the emulsion is produced without refractive index matching of the sample and other materials of the emulsion. 
     
     
         26 . The method of  claim 25 , wherein each droplet of the set of droplets contains one or zero bacterium units of the sample. 
     
     
         27 . The method of  claim 25 , wherein each droplet of the set of droplets has a diameter from 31 microns to 100 microns. 
     
     
         28 . The method of  claim 22 , wherein the sample further comprises a live-dead staining component. 
     
     
         29 . The method of  claim 22 , wherein performing the digital analysis comprises scanning the set of cross-sections of the closed container comprises performing readout of fluorescent signals from individual partitions represented in each of the set of cross-sections. 
     
     
         30 . The method of  claim 29 , wherein scanning the set of cross-sections is performed using light sheet imaging. 
     
     
         31 . The method of  claim 22 , wherein the closed container has a volume from 31 to 100 microliters. 
     
     
         32 . The method of  claim 22 , wherein results of the antimicrobial susceptibility test are returned within less than one day. 
     
     
         33 . A method comprising:
 performing a digital analysis of a set of aqueous droplets generated from a sample and retained at equilibrium positions within a continuous aqueous layer within a closed container, wherein each of the set of aqueous droplets is surrounded by a thin film of oil,   wherein performing the digital analysis comprises scanning a set of cross-sections of the closed container, and wherein the set of aqueous droplets has a number greater than 500,000 droplets.   
     
     
         34 . The method of  claim 33 , wherein performing the digital analysis comprises performing the digital analysis for an antimicrobial susceptibility test of the sample, and wherein the sample comprises bacteria and an antibiotic. 
     
     
         35 . The method of  claim 34 , wherein the sample further comprises a fluorescent growth indicator, and wherein scanning the set of cross-sections of the closed container comprises detecting fluorescent signals from individual droplets represented in each of the set of cross-sections. 
     
     
         36 . The method of  claim 34 , wherein each droplet of the set of droplets contains one or zero bacterium units of the sample, and wherein each droplet of the set of droplets has a diameter from 31 microns to 100 microns. 
     
     
         37 . The method of  claim 33 , wherein performing the digital analysis comprises performing the digital analysis for detection of a cancer form. 
     
     
         38 . The method of  claim 37 , wherein the sample comprises a nucleic acid mixture combined with a solution for polymerase chain reaction (PCR) and a density medium. 
     
     
         39 . The method of  claim 37 , wherein the set of aqueous droplets is characterized by sample occupancy of less than 5%. 
     
     
         40 . The method of  claim 38 , wherein the set of aqueous droplets comprises greater than 22 million droplets, and wherein performing the digital analysis comprises analyzing nucleic acid molecules of the sample without application of Poisson statistics to correct for partitioning error. 
     
     
         41 . The method of  claim 37 , wherein target analytes of the sample comprise cell-free nucleic acids.