Super-resolution fluorescent lifetime imaging method and device based on stimulated emission lost

Abstract

The invention discloses a super-resolution fluorescent lifetime imaging method based on stimulated emission lost. The super-resolution fluorescent lifetime imaging method based on the stimulated emission lost includes the flowing steps: (1) projecting a first laser beam on a to-be-tested sample and activating grains in the to-be-tested sample to an excited sate; (2) utilizing stimulated emission depletion (STED) light to consume the number of the grains which are in the excited state in step (1); (3) utilizing a second laser beam to stimulate the rest grains to emit fluorescent light and collecting the fluorescent light to obtain corresponding fluorescence intensity images; (4) arranging delay between the second laser beam and the STED light, changing delay time, repeating step (3), and obtaining the fluorescence intensity images under different delay time; (5) conducting treatment on the fluorescence intensity images in different delay time through a computer, and fitting a spot intensity attenuation law, inverting lifetime images, and completing scanning of one point of the to-be-tested sample; (6) and completing two-dimension scanning on the sample to be tested by changing the positions where the beam is projected on the to-be-tested sample. The invention further discloses a device used for implementing the method.

Description

technical field [0001] The invention belongs to the field of fluorescence lifetime imaging, in particular to a method and device for long-distance detection of fluorescence lifetime imaging under super-resolution based on stimulated emission loss. Background technique [0002] Fluorescence is what happens when a molecule absorbs energy and its ground state electrons are excited to an excited state and then return from the excited state to the ground state. The fluorescence lifetime refers to the average stay time of the molecule in the excited state before returning to the ground state after being excited by the light pulse. The fluorescence lifetime of the fluorescent substance is generally absolute and is not affected by factors such as the intensity of the excitation light, the intensity of the fluorophore, and photobleaching. It is only related to the structure of the cell itself and its microenvironment. Therefore, fluorescence lifetime imaging microscopy (FLIM), which...

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Problems solved by technology

[0004] However, limited by the optical diffraction limit, the spatial resolution of the traditional fluorescence lifetime imaging method is only about 200 nm, which is difficult to meet the needs of scientific research

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