Single molecule analysis with high accuracy

A process for analysing single molecules, comprising the following steps:

• (a) providing a plurality of single molecules to be analysed, each positioned at an individual sample spot on a support, wherein said spots have a diameter in the range of about 1-20 nm and the distance between individual spots is at least about 10 times, preferably about 20-500 times the diameter of the spot,
• (b) individually illuminating single molecules at individual spots with a light source, wherein said light source provides a plurality of individual illuminated volume elements at the sample spots,
• (c) individually detecting light emitted from said single molecules with a light detector, wherein the light detector comprises a plurality of detection pixels, wherein said detection pixels on the detector have a diameter in the range of about 0.5 μm-50 μm and the distance between said detection pixels is at least about 2 times, preferably about 3-10 times the diameter of the detection pixel, and
• (d) correlating the detected light from an individual detection pixel with an event associated with a single molecule positioned on an individual spot,
• wherein an optical projection of a detection pixel on the support has a diameter in the range of about 100 nm-5 μm and wherein an individual sample spot is aligned to the projection of a single detection pixel on the support, particularly to the center of the projection of a single detection pixel on the support.

A device for analysing at least one individual single molecule, e.g. for sequencing at least one nucleic acid molecule, comprising:

• (a) a support comprising at least one sample spot, wherein said spot has a diameter in the range of about 1 nm-20 nm and the distance between individual spots is at least about 10 times, preferably about 20-500 times the diameter of the spot, for positioning a single molecule to be analysed on an individual spot,
• (b) a light source which provides at least one individual illuminated volume element at a sample spot on the support, for individually illuminating a single molecule at an individual spot,
• (c) a light detector which comprises at least one detection pixel wherein said detection pixel has a diameter in the range of about 0.5 μm-50 μm and the distance between said detection pixels is at least about 2 times, preferably about 3-10 times the diameter of the detection pixel, for individually detecting light emitted from a single molecule at an individual spot, and
• (d) means for correlating the detected signals from individual detection pixels with an event associated with a single molecule positioned on an individual spot,
• wherein an optical projection of a detection pixel on the support has a diameter in the range of about 100 nm-5 μm and wherein an individual sample spot is aligned to the projection of a single detection pixel on the support, particularly to the center of the projection of a single detection pixel on the support.

A device for analysing single molecules, comprising:

• (a) a support comprising a plurality of sample spots, wherein said spots have a diameter in the range of about 1-20 nm and the distance between individual spots is at least about 10 times, preferably about 20-500 times the diameter of the spot, for positioning a single molecule to be analysed on an individual spot,
• (b) a light source which provides a plurality of individual illuminated volume elements at the spots on the support, for individually illuminating single molecules at individual spots,
• (c) a light detector which comprises a plurality of detection pixels, wherein said detection pixels on the detector have a diameter the range of about 0.5 μm-50 μm and the distance between said detection pixels is at least about 2 times, preferably about 3-10 times the diameter of the detection pixel, for individually detecting light emitted from a single molecule at an individual spot, and
• (d) means for correlating the detected light from individual detection pixels with an event associated with a single molecule positioned on an individual spot,
• wherein an optical projection of a detection pixel on the support has a diameter in the range of about 100 nm-5 μm and wherein an individual sample spot is aligned to the projection of a single detection pixel on the support, particularly to the center of the projection of a single detection pixel on the support.