Systems and methods for generation of emulsions with suitable clarity with applications of use
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).
1. A method of counting nucleic acids in a sample, the method comprising:
distributing nucleic acids across a set of droplets of an emulsion within a collecting container, and
upon obtaining cross-sectional images of the collecting container containing the emulsion in a closed configuration of the collecting container, generating a count of only a subset of the set of droplets emitting signals associated with said nucleic acids, with omission of counting of a remainder of the set of droplets.
2. The method of claim 1 , wherein only the subset of the set of droplets contain said nucleic acids.
3. The method of claim 1 , wherein distributing nucleic acids comprises centrifuging sample material containing said nucleic acids, through a substrate comprising a distribution of openings, and into the collecting container at 5% or less occupancy of the set of droplets, and wherein the set of droplets comprises from 1 million to 100 million droplets, such that generating the count omits application of a partitioning error correction.
4. The method of claim 1 , wherein the emulsion is generated upon processing the set of droplets with a set of fluid layers configured with a density gradient across the set of fluid layers, and wherein each fluid layer of the set of fluid layers is immiscible with adjacent fluid layers of the set of fluid layers.
5. The method of claim 4 , wherein the emulsion is generated without refractive index matching of the sample and the set of fluid layers.
6. The method of claim 4 , wherein, upon measuring clarity of the emulsion with a transmission detector, the emulsion is characterized with clarity above a threshold level of clarity along an axis in which measurement of clarity is performed, and wherein the threshold level is 70% optical transmittance of light.
7. The method of claim 1 , wherein generating the count omits implementation of a correction factor for partitioning error.
8. The method of claim 1 , wherein the set of droplets comprises greater than or equal to 1 million partitions.
9. The method of claim 1 , wherein the set of droplets is characterized by less than or equal to 5% occupancy by said nucleic acids.
10. The method of claim 1 , wherein the sample comprises more than 10,000 nucleic acids.
11. The method of claim 1 , further comprising performing prenatal detection of aneuploidy based upon the count.
12. The method of claim 1 , further comprising performing prenatal detection of genetically inherited diseases based upon the count.
13. The method of claim 1 , further comprising performing detection of cancer from cell-free DNA based upon the count.
14. A method of counting analytes in a sample, the method comprising:
distributing analytes across a set of droplets of an emulsion within a collecting container, and
upon obtaining cross-sectional images of the collecting container containing the emulsion in a closed configuration of the collecting container, generating a count of only a subset of the set of droplets emitting signals associated with said analytes, with omission of counting of a remainder of the set of droplets.
15. The method of claim 14 , wherein said analytes comprise one or more of: peptide molecules, protein molecules, and cells.
16. The method of claim 14 , wherein the emulsion is generated upon processing the set of droplets with a set of fluid layers configured with a density gradient across the set of fluid layers, wherein each fluid layer of the set of fluid layers is immiscible with adjacent fluid layers of the set of fluid layers, wherein, upon measuring clarity of the emulsion with a transmission detector, the emulsion is characterized with clarity above a threshold level of clarity along an axis in which measurement of clarity is performed, wherein the threshold level is 70% optical transmittance of light, and wherein the emulsion has clarity above the threshold level of clarity without refractive index matching of the sample and the set of fluid layers.
17. The method of claim 1 , wherein each of the set of droplets has a characteristic diameter from 10 microns to 100 microns, and wherein clarity of the emulsion is produced without refractive index matching of the sample and other fluid components of the emulsion.
18. The method of claim 1 , wherein clarity of the emulsion is produced without refractive index matching of the sample and other fluid components of the emulsion.
19. The method of claim 1 , further comprising performing amplification and labeling of at least part of said nucleic acids within said set of droplets.
20. The method of claim 1 , further comprising providing performing an analysis of single cells of the sample, based upon the counting operation.