Sequencing methods of nucleic acid, single molecule detection methods, how to create the device and sample

摘要

PURPOSE: To detect the position of a single molecule by irradiating a local area of a substrate which has a highly specular surface and to which a sample solution of fluorescent molecules is adhered with exciting light and measuring the frequency of occurrence of a fluorescence intensity. ;CONSTITUTION: After a sample is dissolved in a prescribed solvent and the solution is diluted, the diluted solution is adhered to a substrate 20 in an atomized state and the substrate 20 is naturally dried. A high-purity solvent is used for preparing the solution and the solution is diluted so that the number of molecules which get in an area having an extent nearly equal to the resolution of an optical microscope 42 can become one. Then a luminous area on the substrate 20 is measured while the surface 20 is irradiated with laser light from an exciting light source 30, but the laser light does not get in the visual field of the microscope 42 and the background light becomes extremely small, because the laser light is obliquely made incident to and reflected by the substrate 20. Especially, since the surface of the substrate 20 is finished is a highly specular state and no solvent molecule exists on the surface, the quantity of Raman scattered light becomes smaller and the background light becomes less to such an extent that even fluorescence from one molecule can be caught. Therefore, since a photon counting camera system can measure the intensity of fluorescence and the frequency of occurrence of fluorescence of the intensity at every picture, the position of a single molecule can be detected.;COPYRIGHT: (C)1994,JPO&Japio