Patent ID: 9719928
Date: 2017-08-01
CPC Classifications: G01B,G01N,G02B

Claim:
1. A method of determining the locations of individual molecules of a substance in a sample, wherein the individual molecules of the substance are in a fluorescent state in which they are excitable for emission of fluorescence light by means of excitation light, the method comprising: forming an intensity distribution of the excitation light comprising at least one local minimum of the intensity of the excitation light; exciting the individual molecules of the substance in the fluorescent state for the emission of the fluorescence light by means of the excitation light; registering an intensity of the fluorescence light emitted by the excited individual molecules of the substance in the fluorescent state for different positions of the at least one minimum in an area of interest of the sample; and deducing the locations of the individual molecules of the substance in the fluorescent state from a course of the registered intensity of the fluorescence light over the positions of the at least one minimum in the area of interest of the sample; wherein distances between the individual molecules of the substance in the fluorescent state keep a minimum value d=λ/(2n sin α√(1+I/I wherein the intensity of the fluorescence light emitted by one of the individual molecules at a location of the local minimum of the intensity distribution of the excitation light is at maximum half as high as the intensity of the fluorescence light emitted by the one of the individual molecules at a location of the maximum intensity of the excitation light within the sample; and wherein increments between nearest neighboring positions of the at least one minimum within the sample, in which the fluorescence light from the excited individual molecules of the substance in the fluorescent state is registered, are not greater than half the minimum value d.