Super-resolution fluorescence lifetime correlation spectroscopy system

Abstract

The invention discloses a super-resolution fluorescence lifetime correlation spectrum system. The laser output from laser a is filtered by dichroic filter a and then converged to the microscope objective lens, and the laser output from laser b is filtered by the phase plate and dichroic filter b in turn and then incident on the microscope objective lens; The laser beam converged by the microscope objective lens is irradiated to the sample stage, and the fluorescence signal of the sample to be tested is collected by the microscope objective lens, collected by the collection lens, and then incident on the photodetector, and the photoelectric signal output by the photodetector is input to the optical signal collector ; The synchronous trigger signals of laser a and laser b are input to the optical signal collector. The super-resolution fluorescence lifetime correlation spectroscopy system of the present invention realizes fluorescence lifetime correlation spectroscopy on the basis of super-resolution, reduces the volume of the detection focal spot, and improves the maximum use concentration of the fluorescence lifetime correlation spectroscopy system; due to the use of the fluorescence lifetime separation method , the signal-to-noise ratio of correlation analysis has been greatly improved, and the ability to detect weak signals has been improved.

Description

technical field [0001] The invention relates to a super-resolution fluorescence lifetime correlation spectrum system, which belongs to the field of microscopic imaging. Background technique [0002] Due to the irreplaceable advantages of non-invasive, three-dimensional imaging and specific labeling imaging, optical microscopy has become an important research tool in life sciences today. However, due to the limitation of optical diffraction, traditional optical microscopy can only achieve spatial resolution on the order of wavelength. The rate limits its application in the study of intracellular molecular structure and function at the nanometer scale. Since 2006, researchers from various countries have proposed several new fluorescence imaging principles that break through the diffraction limit, such as photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion (STED) microscopy. Among them, STED mic...

AI Technical Summary

Problems solved by technology

[0007] FLCS is based on confocal microscopy. The area where fluorescence is detected is an Airy disk volume under the optical diffraction limit. The light intensity fluctuations obtained by the FLCS system come from the statistical average of the radiated fluorescence of all fluorescent molecules in the focal spot. Due to Restricted by the diffraction limit, the effective focal spot volume of the confocal microscope is greater than 0.1 femtoliters. In order to obtain a correlation curve with a good signal-to-noise ratio, the average number of molecules in the detection volume should usually be less than 10, so the solution concentration is required to be less than 0.1 micromole per However, many macromolecules in cells are usually dense, which severely limits the application of FLCS in studying the movement of intracellular molecules

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