Device design of high-sensitivity visible-near infrared dual-channel laser fluorescence microscope and imaging method thereof

Abstract

The invention discloses a device design of a high-sensitivity visible-near infrared dual-channel laser fluorescence microscope and an imaging method thereof. The device comprises a visible light path(A) and a near-infrared light path (B); the imaging method comprises the steps of mechanical calibration, fine adjustment by using Photoshop software and image fusion by using Image-J software. According to the method, visible light and near-infrared fluorescence in the same microscopic region can be observed, and convenient switching is realized. The monochromaticity of the laser is high, and theexcitation energy is gathered along with the increase of the multiplying power; meanwhile, the filtering efficiency of the optical filter combination on background stray light and excitation light ishigh, and the detection sensitivity on fluorescent substances can be improved. According to the invention, visible and near-infrared fluorescence double-channel observation of an ultrathin transparent biological sample is realized, near-infrared fluorescence single-channel observation can be carried out on the surface of an object with any thickness, and an intuitive means is provided for microscopic distribution observation of near-infrared fluorescent substances (especially indocyanine green and derivatives thereof).

Description

technical field [0001] The invention belongs to the field of fluorescence microscopy technology, and in particular relates to a high-sensitivity device design and imaging method for visible-near-infrared dual-channel imaging using laser excitation. Background technique [0002] As an important tool in the current biomedical field, bioluminescent imaging technology can perform real-time and visualized detection of biological components, cells and tissues / organs on a three-dimensional scale. It has been widely used in many fields such as tumor diagnosis, biomolecular detection, drug distribution and metabolism, etc. Fluorescence microscopy imaging technology can detect the structure and composition information of the surface or interior of the sample by detecting the spatial distribution of fluorescent substances in the sample, especially in the microscopic detection of fluorescent labels in biomedicine. The fluorescence detection of most fluorescence microscopes is limited t...

AI Technical Summary

Problems solved by technology

This type of method has slow imaging speed and large data flow, which requires powerful computer analysis and processing capabilities; the image reconstructed by the analog signal is not a real optical signal, which distorts the spatial distribution of the fluorescent signal; long-term laser excitation may cause fluorescence molecular quenching. destroy or even degrade, changing the information of the sample itself

In addition, the self-made near-infrared fluorescence microscope that can be directly imaged in the laboratory uses LED as the excitation light source. The poor monochromaticity leads to low excitation efficiency. Usually, there are only 1 or 2 fluorescence filters, which leads to insufficient light splitting efficiency of the optical path. The sensitivity and specificity of substance detection are not high

And there is still a lack of powerful equipment for microscopic observation of near-infrared fluorescent substances in China, so it is very necessary to develop a visible-near-infrared dual-channel laser fluorescence microscope

Images