Methods and systems for monitoring reactions
Methods and systems for monitoring reactions by observing signals deriving from those reactions, using signal processing that allows differentiation between signals that are otherwise optically overlapping by conventional detection methods. Centroid determination is used to identify signal sources that are presenting confounding overlapping signals due to their physical proximity, and/or to identify discrete signals from different reaction centers.
1. A method of monitoring single molecule reactions at a first and a second single molecule reaction complex, comprising:
providing a first single molecule reaction complex and a second single molecule reaction complex, each at least partially immobilized on a substrate,
exposing the first and second single molecule reaction complexes to reaction conditions;
wherein said first single molecule reaction complex produces a first optically detectable signal indicative of the presence of a single molecule reaction at the first single molecule reaction complex and the second single molecule reaction complex produces a second optically detectable signal indicative of the presence of a single molecule reaction at the second single molecule reaction complex, wherein the first and second optically detectable signals are not completely optically resolvable from each other;
positionally detecting the first optically detectable signal and the second optically detectable signal;
identifying a first positional centroid for the first optically detectable signal and a second positional centroid for the second optically detectable signal; and
characterizing the first optically detectable signal as emanating from the first single molecule reaction complex based upon the first positional centroid and characterizing the second optically detectable signal as emanating from the second single molecule reaction complex based upon the second positional centroid, and;
detecting a plurality of optically detectable signals from the first and second single molecule reaction complexes and assigning each of said plurality of signals as emanating from either the first or second single molecule reaction, complex based upon a comparison of a positional centroid of each of the plurality of optically detectable signals with a positional centroid determined from the first and second single molecule reaction complexes.
2. The method of claim 1 , wherein the identification of the first positional centroid is determined at the sub-pixel level.
3. The method of claim 1 , wherein the first single molecule reaction complex comprises a nucleic acid polymerase/template/primer complex.
4. The method of claim 3 , wherein the first optically detectable signal indicative of the presence of the first single molecule reaction complex comprises a fluorescent signal indicative of polymerase mediated extension of a primer in the complex to produce an extended primer.
5. The method of claim 4 , wherein the first optically detectable signal comprises a fluorescent label on a labeled nucleotide or nucleotide analog that has been incorporated into or is being incorporated into the extended primer.
6. The method of claim 5 , wherein the labeled nucleotide is labeled upon a portion of the nucleotide that is retained upon the nucleotide following incorporation.
7. The method of claim 5 , wherein the labeled nucleotide is labeled upon a portion of the nucleotide that is not retained upon the nucleotide following incorporation.
8. The method of claim 1 , wherein the first and second optically detectable signals comprise substantially the same spectrum.
9. The method of claim 1 , wherein the first and second optically detectable signals comprise different spectra.
10. The method of claim 1 , wherein the first single molecule reaction complex comprises a nucleic acid polymerase/template/primer complex, and the first optically detectable signal comprises a fluorescent signal emanating from a nucleotide that has been or is being incorporated into a primer extension reaction by the polymerase, the optically detectable signal being indicative of a type of nucleotide that is incorporated or being incorporated.
11. The method of claim 1 , wherein the first and second optically detectable signals are temporally distinct.