Super-resolution fluorescence microscopy method and device based on tangential polarization

Abstract

The invention discloses a super-resolution fluorescence microscopy method based on tangential polarization, comprising the following steps: carrying out 0-2pi vortex phase coding focus on tangential polarization exciting light, and obtaining an exciting spot below a diffraction limit on a fluorescence sample; adjusting tangential polarization STED laser and the phase coding tangential polarization exciting light to realize confluence and co-axis, focusing on the fluorescence sample to form a circle bread-shaped focusing spot the central point of which coincides with the central point of the exciting spot; adjusting the operating power of the STED laser to cause the area of a luminous point in the exciting spot to reach the super-resolution ratio; and collecting fluorescence emitted from the luminous point and carrying out detection processing to obtain a microimage with the super-resolution ratio. The invention also discloses a device for realizing the super-resolution fluorescence microscopy method based on tangential polarization. In the invention, on the premise of ensuring the super-resolution ratio, the working power of the STED is reduced greatly, thereby lowering bleaching of the sample and avoiding damage for the sample.

Description

technical field [0001] The invention relates to the field of fluorescence microscopy, in particular to a super-resolution fluorescence microscopy method and device. Background technique [0002] Since Abbe first discovered the phenomenon of diffraction limitation in far-field optical imaging in 1873, it has been recognized in the physics community that the resolution of far-field optical microscopy is limited by the optical diffraction limit, which is related to the wavelength and numerical aperture of the light source. Due to the limitation of optical diffraction, it is impossible to obtain an ideal image point by imaging an ideal point object through an optical system, but a Fraunhofer diffraction image. The general optical system is circular, and the Fraunhofer diffraction image is the so-called Airy disk. The image of each object point on the image plane is a diffuse spot, and it is difficult to distinguish two diffuse spots when they are close together, which limits th...

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Problems solved by technology

(4) The theoretically infinite resolution is actually limited, because the pursuit of high resolution also increases the fluorescence bleaching, thereby reducing the imaging resolution and imaging quality

Its characteristics: first, it is difficult to focus circularly polarized excitation light below the diffraction limit; second, use circularly polarized light to pass through a vortex phase plate to form a doughnut to suppress the fluorescence around the excitation spot, and the suppression effect can only be achieved by increasing the STED laser power

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