Compositions and methods for disease diagnosis using single cell analysis
Certain embodiments of the invention are directed to evaluating and identifying cells by recording and interpreting a time-dependent signal produced by unique cell respiration and permeability attributes of isolated viable cells.
1 . A method for identifying a cell comprising:
(a) contacting a sample containing cells with a first reporter that is a pH indicating fluorophore, thereby forming a sample mixture;
(b) partitioning the sample mixture into a plurality of volume partitions containing one cell or one cellular aggregate;
(c) incubating the volume partitions for a period of time at a specified temperature or series of temperatures;
(d) monitoring optical characteristics of the volume partitions during the incubation time;
(e) constructing waveforms for the volume partitions based on the optical characteristics over time; and
(f) identifying the cell in the volume partition based on the waveform constructed for the volume partition.
2 . The method of claim 1 :
wherein forming the sample mixture further includes contacting the sample containing cells with resorufin; and
wherein the optical characteristics include optical characteristics of the fluorophore and the resorufin.
3 . The method of claim 1 , wherein the volume partitions are droplets in an immiscible fluid.
4 . The method of claim 3 , wherein the immiscible fluid is a fluorocarbon comprising a fluorosurfactant.
5 . The method of claim 3 , wherein the sample is partitioned by Laplace pressure gradients.
6 . The method of claim 3 , wherein the sample is partitioned by shear stress.
7 . The method of claim 1 , further comprising arranging the volume partitions in a two-dimensional array.
8 . The method of claim 7 , wherein the two-dimensional array is a static two-dimensional array.
9 . The method of claim 1 , wherein monitoring the optical characteristics of the volume partitions is performed using a camera.
10 . The method of claim 1 , wherein the identification of the cell using the waveform includes comparing the volume partition waveform to a library of stored waveforms.
11 . The method of claim 1 , wherein forming the sample mixture further includes contacting the sample containing cells with a second reporter; and
wherein the emission spectra of the fluorophore and the second reporter are different.
12 . The method of claim 11 , wherein the second reporter is a two-part molecule comprising (a) a protein and (b) a molecule selected from the group consisting of a photooxidizable cofactor, a mitochondrial vital stain or dye, a dye exhibiting a redox potential, a membrane localizing dye, a dye with energy transfer properties, a pH indicating dye, a resazurin dye, a tetrazolium dye, a coumarin dye, an anthraquinone dye, a cyanine dye, an azo dye, a xanthene dye, an arylmethine dye, a pyrene derivative dye, a ruthenium bipyridyl complex dye, and a derivative of one of the aforementioned dyes.