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 comprising:
performing a digital analysis of a sample distributed across a set of partitions,
wherein performing the digital analysis comprises imaging a set of cross-sections of a closed container containing the set of partitions, wherein the set of partitions has a number greater than 1 million partitions, and wherein the digital analysis is performed within three minutes.
2. The method of claim 1 , wherein the digital analysis is performed within two minutes.
3. The method of claim 1 , wherein imaging the set of cross-sections of the closed container comprises performing readout of signals from contents of partitions represented in each of the set of cross-sections.
4. The method of claim 3 , wherein the signals comprise fluorescent signals from labelled target analytes of the sample.
5. The method of claim 1 , wherein the set of partitions comprises a set of droplets of an emulsion generated from the sample, wherein clarity of the emulsion is produced without refractive index matching of the sample and other materials of the emulsion.
6. The method of claim 5 , wherein each of the set of droplets has a characteristic diameter from 10 microns to 100 microns.
7. The method of claim 5 , wherein the set of droplets is characterized by less than 5% occupancy by target analytes of the sample.
8. The method of claim 1 , wherein the closed container has a volume from 10 to 100 microliters.
9. The method of claim 1 , wherein performing the digital analysis comprises analyzing up to 1.4 million nucleic acid molecules of the sample without application of Poisson statistics to correct for partitioning error.
10. The method of claim 1 , wherein the sample comprises a nucleic acid mixture combined with a solution for polymerase chain reaction (PCR) and a density medium.
11. The method of claim 10 , wherein the nucleic acid mixture comprises cell-free nucleic acids.
12. The method of claim 1 , wherein the sample comprises material derived from fetal cells and maternal cells, and wherein performing the digital analysis further comprises performing prenatal detection of aneuploidy.
13. The method of claim 1 , wherein the sample comprises a protein solution.
14. The method of claim 1 , wherein imaging the closed container comprises performing a 3D scanning technique.
15. The method of claim 1 , wherein imaging the closed container comprises performing a planar imaging technique.
16. A method comprising:
performing a digital analysis of a set of droplets generated from a sample and retained at equilibrium positions within a continuous fluid layer within a closed container,
wherein performing the digital analysis comprises scanning one or more cross-sections of the closed container, wherein the set of droplets has a number greater than 1 million droplets, and wherein the digital analysis is performed within three minutes.
17. The method of claim 16 , wherein each of the set of droplets has a characteristic diameter greater than 1 micron.
18. The method of claim 16 , wherein scanning one or more cross-sections comprises performing at least one of a 3D scanning technique and a planar imaging technique.
19. The method of claim 16 , wherein:
the sample comprises a first aqueous solution comprising target nucleic acids, a mixture for polymerase chain reaction (PCR), and a density medium,
the continuous fluid layer comprises a second aqueous solution, and
the set of droplets generated from the sample are individually isolated and separated from the second aqueous solution by a non-aqueous solution.
20. The method of claim 19 , wherein performing the digital analysis further comprises performing a temperature adjustment operation on the set of droplets at the equilibrium positions, wherein the set of droplets is thermally stable during the temperature adjustment operation.
21. A method comprising:
performing a digital analysis of a set of droplets generated from a sample and retained at equilibrium positions within a continuous fluid layer within a closed container,
wherein performing the digital analysis comprises scanning a set of cross-sections of the closed container,
wherein the set of droplets comprises 3.5 million droplets,
wherein the digital analysis is performed within three minutes, and
wherein performing the digital analysis comprises performing prenatal detection of a genetic disorder.