A rotating total internal reflection microscopy method and device with feedback

Abstract

The invention discloses a rotating total internal reflection microscopy device with a feedback function. The device comprises a laser device, a two-dimensional scanning galvanometer, a scanning lens, a collimating lens, a dichroic mirror, a beam splitter, a dichroic mirror, a microscopic field lens, a total reflection microobjective and a sample which are arranged in sequence along an optical path, a light intensity position detector located on the reflection optical path of the beam splitter, a computer and a CCD used for acquiring the sample and emitting fluorescent light, wherein the light intensity position detector is used for collecting illuminating light which is emitted by the laser device and reflected by the beam splitter to obtain a first light spot and collecting sample light formed after total reflection of the position of the sample to obtain a second light spot, and the computer is used for obtaining total reflection illumination angle and evanescent wave penetration depth through feedback according to the position information of the first light spot and the second light spot. The invention further discloses a rotating total internal reflection microscopy method with the feedback function. Through synchronous feedback control of the sample and the microobjective, it can be guaranteed that the sample is located on the optimal illumination face and the optimal imaging face, and illumination uniformity and imaging resolution are higher.

Description

technical field [0001] The invention relates to the field of microscopic imaging, in particular to a rotating total internal reflection microscopic method and device with feedback. Background technique [0002] With the development of biological science research, especially the study of membrane-related biological phenomena, there is a strong demand for a microscopic method that only observes the structure of single-layer samples. In the traditional microscopy method, the entire field of view is illuminated by the illumination beam in the z-axis direction during illumination, and the resolution and signal-to-noise ratio in the z-axis direction have not been high. For this reason, light sectioning microscope and total internal reflection microscope ( Tirf) are technical means to improve the z-axis resolution. [0003] Light section microscope adopts the method of lateral illumination, but due to the existence of the diffraction limit, the thinnest beam of lateral illuminatio...

AI Technical Summary

Problems solved by technology

However, due to the existence of laser speckle, the illumination light field will be uneven. For this reason, a rotating total internal reflection microscopy method can overcome this shortcoming and achieve uniform total reflection light field illumination. In the microscopic method of rotating total reflection illumination , the lighting angle determines the lighting depth, but it belongs to an open-loop control in the adjustment process, one is to judge the lighting angle according to the imaging situation, but the control accuracy of the lighting angle is not enough in this process, and it cannot be adjusted in real time

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