Tunable Penetration Depth Tirf Microscope and Method Based on Ellipsoidal Mirror

Abstract

The invention discloses an ellipsoid reflector-based penetrating depth adjustable TIRF microscope and a method therefor and belongs to the field of optical wide field microscopic imaging. The microscope consists of a cone reflector-based annular parallel light beam generation optical path, an ellipsoid reflector-based illumination optical path and a common fluorescence microscopic imaging optical path; according to the method, sample excitation depth can be changed via adjustment of distance between two complementary cone reflectors. According to the penetrating depth adjustable TIRF microscope and the method therefor, omnibearing shadow-free illumination is provided for samples based on rotation characteristics of an ellipsoid reflector, lighthouse-shaped flaws caused by unidirectional illumination in a conventional solution can be eliminated, and imaging quality can be improved; based on a characteristic of a large numerical aperture of the ellipsoid reflector, total internal reflection is provided in such a way that a sample illumination incidence angle can be precisely and finely adjusted in a large scope ranging from a critical angle of total reflection to nearly a 90 degree angle, requirements of gradually rising TIRF axial super-resolution three dimensional imaging research for finely adjustable sample excitation depth can be met, and finally TIRF imaging quality and axial resolution can be greatly improved.

Description

technical field [0001] The ellipsoid reflector-based TIRF microscope with adjustable penetration depth of the invention belongs to the field of optical wide-field microscopic imaging and the field of optical TIRF imaging technology, and can be widely used in research in the fields of life science, physical chemistry, material science and the like. Background technique [0002] TIRF microscope uses the evanescent wave generated near the medium interface after total reflection of light to excite the fluorescence of the sample. Since the intensity of the evanescent wave decays exponentially along the direction perpendicular to the interface, only a small thin layer of several hundred nanometers near the interface The sample area can be excited, which greatly reduces the interference of background light noise, so this technology is widely used in technical fields that require high signal-to-noise ratio, especially suitable for studying the dynamic imaging process of single molecu...

AI Technical Summary

Problems solved by technology

However, most of the existing devices are based on the objective lens type TIRF. On the one hand, the incident angle range of the illumination light is limited by the structure of the objective lens, and the maximum does not exceed 78 degrees. On the other hand, due to the need to adjust the incident angle of the illumination light, the device structure is relatively complicated. Therefore, the schemes adopted are all one-way lighting. At this time, the defects caused by lighting shadows are introduced, and the existing structures for adjusting the incident angle of lighting light are costly, such as using scanning galvanometer systems, acousto-optic modulation systems, etc.

These unfavorable factors are not conducive to the good TIRF imaging effect and the research and development of related technologies.

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