SYSTEMS AND METHODS FOR GENERATING DROPLETS AND PERFORMING DIGITAL ANALYSES
The disclosure provides compositions, methods, and systems for implementation of high-performance molecular diagnostic assays, where generation of counts of targets from a sample is achieved in a rapid manner, with respect to sample partitioning and target detection, in the single-molecule regime.
1 . A method comprising:
(a) providing a plurality of partitions, wherein the plurality of partitions is within a continuous phase within a container;
(b) using an imaging system to scan the plurality of partitions across a set of optical channels to detect signals from at least a subset of the plurality of partitions; and
(c) using the signals detected in (b) to generate a count of a number of the target molecules.
2 . The method of claim 1 , wherein the target molecules comprise nucleic acid molecules.
3 . The method of claim 1 , wherein the plurality of partitions comprises a set of wells.
4 . The method of claim 1 , wherein (b) comprises scanning a sheet of the container using a light sheet imaging system.
5 . The method of claim 1 , wherein the plurality of partitions comprises at least 1 million partitions.
6 . The method of claim 1 , wherein the plurality of partitions comprises at least 5 million partitions.
7 . The method of claim 1 , wherein (c) is performed within 5 seconds.
8 . The method of claim 1 , wherein the plurality of partitions comprises a plurality of nucleic acid molecules.
9 . The method of claim 8 , further comprising before (b), amplifying the plurality of nucleic acid molecules.
10 . The method of claim 1 , wherein (c) comprises identifying partitions of the set of partitions comprising the target molecule based on satisfaction of an optical property criterion, a shape criterion, and a morphology criterion.
11 . The method of claim 10 , wherein the optical property criterion is based on a lateral or axial intensity gradient across a set of pixels.
12 . The method of claim 10 , wherein the shape criterion is based on a radius of a partition of the set of partitions.
13 . The method of claim 10 , wherein the morphology criterion is based on a three-dimensional profile of a partition profile of a control sample.
14 . The method of claim 10 , wherein:
i) wherein the optical property criterion is based on a lateral or axial intensity gradient across a set of pixels;
ii) wherein the shape criterion is based on a radius of a partition of the set of partitions; and
iii) wherein the morphology criterion is based on a three-dimensional profile of a partition profile of a control sample.
15 . The method of claim 1 , wherein the count of the number of target molecules is between 1 target molecule and 1 , 000 , 000 target molecules.
16 . The method of claim 10 , wherein identifying partitions of the set of partitions comprising the target molecule is further based on satisfaction of a signal intensity criterion.
17 . The method of claim 16 , wherein the signal intensity criterion is based upon maximum convoluted intensity of the set of pixels, a difference between the maximum intensity and the minimum intensity of the set of pixels, and a total intensity of the set of pixels.
18 . The method of claim 16 , wherein the signal intensity criterion is based upon a maximum signal intensity of the set of pixels.
19 . The method of claim 1 , wherein the container has a volumetric capacity of 50 microliters.
20 . The method of claim 1 , wherein the container is a tube.