Three-channel fluorescence positioning super-resolution biological microscopy system and microscopy method

Abstract

The invention discloses a three-channel fluorescence positioning super-resolution biological microscopic system and a microscopic method. The three-channel fluorescence positioning super-resolution biological microscopic system comprises a microscope main body, an illumination module, an imaging module, a three-dimensional nanoscale sample locking module and a control and display module in signalconnection; in an imaging process, three-channel laser illumination light irradiates a sample at the same time; three types of fluorescent dyes for marking the sample emit light and flicker at the same time; through the imaging module, imaging at different parts of the detector is realized; the detector collects original images with a plurality of fluorescent dye flicker signals, and transmits theoriginal images to the control and display module, and the control and display module finds out the central position of each flicker point in each original image through Gaussian fitting, and superposes the central positions of the flicker points of all the original images together to obtain ultrahigh-resolution images of three channels. According to the system and the method, the problem of drifting of the sample in a three-dimensional space is solved, the imaging time can be shortened by two times, and three-channel simultaneous imaging is ensured to be realized.

Description

technical field [0001] The invention relates to the field of biological microscopy, in particular to a three-channel fluorescence positioning super-resolution biological microscopy system and a microscopic method. Background technique [0002] Since Ernst Abbe (Ernst Abbe) put forward the theory of the resolution limit of optical imaging in the 1870s, people have been looking for various ways to break through this resolution limit. At present, through the use of modern cutting-edge technology, Zhuang Xiaowei and Eric Betzig (Eric Betzig) respectively proposed Stochastic Optical Reconstruction Microscopy (STORM) and Fluorescence Localization Microscopy in 2006. (Photoactivation Laser Microscopy, PALM), have achieved super-resolution imaging that breaks through the optical resolution limit by ten times. Eric Betzig won the 2014 Nobel Prize in Chemistry for this technique. At present, fluorescence localization microscopy has been partially commercialized and has begun to be u...

AI Technical Summary

Problems solved by technology

Although this drift phenomenon generally exists in microscopic systems, the phenomenon caused by drift is not obvious because the resolution of general microscopes is less than 300 nanometers and the time required for imaging is relatively short.

The disadvantage of using the algorithm to correct the drift is that the algorithm cannot be used for samples with unclear structures, and it can only correct the linear drift of the sample, but it cannot do anything about the nonlinear drift; the defect of using fluorescent particles for drift correction is that it is troublesome to prepare and it is impossible to determine whether the fluorescent particles have Fixed in the sample, in addition, due to photobleaching, the fluorescence of fluorescent particles will decay with imaging time, so the accuracy of drift correction will also deteriorate with time

[0006] On the longitudinal Z axis, the current method of controlling sample drift is to use a beam of infrared or ultraviolet light to obtain the displacement through reflection interference, and actively move the objective lens to compensate for the displacement of the sample. The defect of this method is that it cannot image deep cells. And if the sample radiates infrared or ultraviolet light, it will affect the operation of the locking program

[0007] Secondly, it is difficult to realize multi-channel super-resolution fluorescence microscopy. If the method of sequential imaging is used to realize multi-channel, the multi-channel is sequentially excited with different wavelengths of lasers to separately excite fluorescent dyes for imaging. The problem is that the imaging time is too long , the efficiency is low and the sample drift is not easy to control; the ultra-high imaging laser intensity is strong, when imaging one channel, wrong excitation of other channels will lead to the quenching of the fluorescent dye of the labeled sample, resulting in insufficient labeling and affecting the imaging quality; and different channels There will be a certain degree of crosstalk between them, which will affect the imaging accuracy

[0008] Finally, STORM and PALM imaging require the combination of fluorescent dye molecules and imaging buffer components to produce a "blinking" effect under strong laser irradiation. High-quality "blinking" requires fluorescent molecules to stay in the bright state for a long time. Short, but the number of photons emitted is large, and different fluorescent dye molecules have different requirements for the composition of the buffer when they can "blink" with high quality, that is, the more channels of super-resolution imaging, the search for fluorescent dye molecules Compatibility with imaging buffer components is more difficult

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