Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere

Abstract

The invention discloses a method and device for analyzing the fluorescent correlation spectroscopy based on a medium microsphere. In the method, radial polarized light and tangential polarized light are respectively used as a fluorescent excitation beam and a fluorescent inhibition beam which are sequentially focused by a microobjective, then are subjected to nanometer spraying by virtue of the medium microsphere and then act on a fluorescent sample and excite a fluorescent signal; and fluorescent correlation spectroscopy analysis is completed by collecting and analyzing the fluorescent signal. The device sequentially comprises a light source generating the radial polarized light and the tangential polarized light, a first microobjective, the medium microsphere, a sample frame provided with the fluorescent sample and a second microobjective in sequence as well as a fluorescent signal analysis processing device connected with the second microobjective; the first microobjective, the medium microsphere, the fluorescent sample and the second microobjective are located on a coaxial optical path of the radial polarized light and the tangential polarized light; and the medium microsphere is arranged on the object focal plane of the first microobjective. The method and device provided by the invention can be effectively applied to a high-concentration fluorescent molecule sample.

Description

technical field [0001] The invention belongs to the field of optical metrology and super-resolution imaging, and in particular relates to a fluorescence correlation spectrum analysis method and device based on medium microspheres. Background technique [0002] The development of science and technology has greatly expanded people's horizons. With the deepening of research, people have become more and more interested in the microscopic field. In many fields such as chemistry, biology, and medicine, in order to more accurately grasp the properties of the research object, the detection and analysis of single molecules has become an indispensable research method. In order to meet the above requirements, a large number of corresponding instruments and equipment have been developed, and the Fluorescence Correlation Spectroscopy (FCS) is one of them. By dynamically recording the intensity of the collected fluorescent signals and performing self-correlation digital signal processin...

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Problems solved by technology

[0003] However, as an optical confocal imaging system, the resolving power of FCS is inevitably limited by the optical far-field diffraction limit.

Therefore, when the concentration of fluorescent molecules in the test sample is too large, it is often impossible to perform single-molecule measurement effectively and interfere with the final test results.

The most direct way to solve this problem is to dilute the test sample. However, in view of the fact that many biochemical reactions must be completed in a high-concentration infiltration environment, the test data obtained by the dilution method is not reliable, and may even be impossible due to the reaction. resulting in a test failure

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