Nonlinear Structured Illumination Microscopic Imaging Method and System

Abstract

The invention discloses a non-linear structure light illumination microscopic imaging method which comprises the following steps: 1) loading a computed hologram on a digital microscopic array; 2) generating a first spatial structure light field which meets sine distribution and is used for activating fluorescent protein, and radiating the first spatial structure light field to the surface of a sample, so as to convert a part of protein to be in an illuminated state from a dark state; 3) radiating the sample in a second spatial structure light field so as to enable fluorescent protein in the bright state to emit fluorescent light, collecting the fluorescent light, and imaging in a photoelectric detector; 4) repeating the step 2) and the step 3), acquiring a plurality of spatial frequencies, acquiring a plurality of initial phases in each direction to obtain a plurality of original images, and reestablishing a super-resolution image according to a GPU acceleration algorithm. The invention further discloses a non-linear structure light illumination microscopic imaging system. The non-linear structure light illumination microscopic imaging method has the advantages of relatively high system imaging resolution, high fluorescent drifting resistance, low phototoxicity and rapid imaging.

Description

technical field [0001] The invention relates to the field of biological imaging, in particular to a non-linear structured illumination microscopic imaging method and system. Background technique [0002] Bioimaging is a frontier field of life science involving multiple disciplines. It is a cutting-edge imaging technology for studying biological functions by "seeing" the interaction of living cells in real time and dynamically at different levels such as molecules, cells, tissues, and living bodies. It has become a breakthrough point for major achievements in life sciences today. [0003] The traditional optical microscope is limited by the optical diffraction limit. The size of the diffraction spot (Airy disk) is about half of the ratio of the wavelength of the light source to the numerical aperture of the microscope objective lens. Generally, the highest imaging resolution is around 200-300nm, and it is impossible to directly observe some How important molecules such as ge...

AI Technical Summary

Problems solved by technology

In 2005, Gustafsson.etal. [PNAS, 102: 13801 (2005)] proposed a nonlinear structured light illumination super-resolution imaging method (NL-SIM), which can obtain high-frequency information of samples by using the saturated nonlinear characteristics of fluorescent molecules , in theory, any high-resolution fluorescence imaging can be achieved. However, in order to achieve saturated nonlinear fluorescence emission, a high excitation light power density is required, which is easy to cause damage to biological tissues. At the same time, NL-SIM needs to collect data that is several times that of SIM. In order to reconstruct a super-resolution image, the imaging speed is slow, these problems limit the practical application of NL-SIM in the field of biomedicine

Images