Single-wavelength realization of multiphoton pulsed sted-spim microscopy system

Abstract

The invention relates to a multiphoton subpulse STED-SPIM (Selective Plane Illumination Microscopy) microscopic system realized by single wavelength, characterized in that the femtosecond pulse laser emitted by a femtosecond single wavelength laser is transmitted to a first splitter through a first polarization regulator to be divided into two beams of light, and a first beam of light is successively transmitted to a second polarization regulator, a second splitter, a third polarization regulator and a quarter-wave plate along a Y-axial direction to form an excitation light beam; a second beam of light is transmitted to an STED photoquenching system, and the light outputted by the STED photoquenching system is successively emitted into the second splitter, the third polarization regulator and the quarter-wave plate to be modulated into donut-shaped focus spots to form a quenching light beam; a scanner is used for scanning the excitation light beam and the quenching light beam to generate an excitation plate light source and an STED quenching plate light source; an excitation plate light source and an STED quenching plate irradiate an object to be imaged through an excitation objective lens to allow the flourescent dye of the object to be imaged to excite fluorescence, and excited fluorescence is imaged through an imaging objective lens, and is emitted to a photosensitive element or an analysis element successively through an optical filter and a convergent lens.

Description

technical field [0001] The invention relates to a single-wavelength multi-photon pulse STED-SPIM microsystem, which belongs to the technical field of biomolecular imaging. Background technique [0002] Multiphoton microscopy is a new technology that combines laser scanning confocal microscopy and multiphoton excitation technology. The basic principle of multi-photon excitation: in the case of high photon density, fluorescent molecules can absorb two long-wavelength photons at the same time, and the energy of these two photons can be added to make the electrons of fluorescent molecules jump to an excited state. Exciting a fluorescent molecule with a photon whose wavelength is half the long wavelength is comparable. Multiphoton microscopy has many advantages: 1) small photobleaching area for samples; 2) low phototoxicity; 3) strong penetrating power, the penetration depth of multiphoton microscope is usually 2 to 3 times that of confocal microscope; 4) Imaging brightness and...

AI Technical Summary

Problems solved by technology

Adjust the three-dimensional space coincidence of laser and STED light, in which STED is adjusted into a doughnut-shaped spot through a phase plate and a quarter-wave plate, and the energy level structure of fluorescent molecules and stimulated radiation are used to selectively consume excited fluorescent molecules in the edge region of PSF. The PSF scale is compressed, and the final fluorescent signal reaches the APD (avalanche photodiode) through two dichroic mirrors. In theory, the resolution can be infinitely improved with the increase of the STED light intensity. However, STED is usually a point scan, so its imaging speed is relatively slow , the field of view is small, and STED requires two lasers for excitation and quenching, and the cost is high

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