Super-resolution microscopic method and device based on fluorescence lifetime difference

Abstract

The invention discloses a super-resolution microscopic method based on fluorescence lifetime difference. The super-resolution microscopic method comprises the following steps: (1) focusing an excitation beam and a suppressed beam by a large numerical aperture microscope objective and scanning a fluorescence sample is scanned simultaneously by taking circularly polarized light as the excitation beam and taking the circularly polarized light coded by vortex phase as the suppressed beam; (2) collecting fluorescence emitted by the fluorescence sample by utilizing the large numerical aperture microscope objective and obtaining a view picture of fluorescence intensity image by a photoelectric sensing device; (3) obtaining corresponding fluorescence lifetime information by analyzing the fluorescence intensity information of the fluorescence intensity image; (4) setting a time gate for separating a long-life fluorescence image and a short-life fluorescence image from the fluorescence intensity information; and (5) setting a weight and subtracting the weighted long-life fluorescence image from the short-life fluorescence image to obtain a final super-resolution microscopic image. The invention also discloses a super-resolution microscopic device based on the fluorescence lifetime difference.

Description

technical field [0001] The invention relates to the field of microscopic imaging, in particular to a super-resolution microscopic method and device based on fluorescence lifetime difference. Background technique [0002] Like most optical imaging, the Abbe diffraction limit has restricted the improvement of the resolution of the microscope system since the invention of the microscope. Early microscope systems were all wide-field imaging systems with limited imaging resolution. This situation was not improved to some extent until the invention of the confocal microscope system (Confocal Microscope). The basic concept of confocal microscopy was proposed by M.Minsky et al. in 1957 (see M.Minsky et al. Microscopy Apparatus, US Patent 3013467), but the technique was not really instrumented until 1978 (see C.Cremer et al. Considerations on a laser-scanning-microscope with high resolution and depth of field, Microscopia Acta 81, 31-44 (1978)). Compared with the traditional wide-...

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Problems solved by technology

However, this method reduces the intensity of the fluorescent signal that can be effectively used, reduces the signal-to-noise ratio of the system, and easily has an adverse effect on the actual resolution capability of the system.

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