Two-photon fluorescence microscopy method and device based on photon recombination

Abstract

The invention discloses a two-photon fluorescence microscopy device based on photon recombination. The device comprises a femtosecond laser device, an optical parameter oscillator, a first half wave plate, a polarization beam splitter, a second half wave plate, a first quarter wave plate, an objective lens, an array detector and a computer, wherein the femtosecond laser device is used for sending ultra-short pulse laser to perform two-photon excitation on samples; the first half wave plate is used for regulating the proportion of light s and light p of emergent light of the optical parameter oscillator; the polarization beam splitter is used for regulating the light intensity; the second half wave plate is used for regulating the polarization direction of the line polarization; the first quarter wave plate is used for converting the line polarization light into round polarization light; the objective lens is used for focusing the round polarization light into solid round spots for illuminating a sample and exciting the fluorescence; the array detector consists of a plurality of detector units, and is used for collecting fluorescence signals; the computer processes the fluorescence signals obtained by each detector unit to obtain an image corresponding to one object point. The invention also discloses a two-photon fluorescence microscopy method based on photon recombination. The detector array is used for replacing the original single needle hole detector, so that the obtained image has a higher signal-to-noise ratio.

Description

technical field [0001] The invention belongs to the field of confocal microscopy, in particular to a two-photon fluorescence microscopy method and device based on photon reconstruction. Background technique [0002] Two-photon fluorescence microscopy enables three-dimensional imaging in living biological samples. Compared with traditional confocal microscopy, two-photon can achieve better optical sectioning function, deeper penetration depth and less phototoxicity. The single-photon excitation process is that the ground state electron absorbs a short-wavelength photon to reach the excited state. This process generally requires ultraviolet or blue / green light, and these short-wavelength light may cause severe phototoxicity to biological samples. For two-photon excitation, under the condition of sufficient light intensity, the excitation of ground state electrons can be realized by simultaneously absorbing two photons with lower energy (the wavelength is usually in the infra...

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Problems solved by technology

However, structured light microscopy is generally used in wide-field microscopy. Although stimulated emission depletion microscopy can be applied to two-photon microscopy, it requires high alignment accuracy.

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