Dual-color two-photon fluorescence imaging method and device

Abstract

The invention provides a dual-color two-photon fluorescence imaging method and a device, which uses six dual-color beams for emitting along plus and minus X, plus and minus Y, plus and minus Z six directions into the sample 3, thereby forming a three-dimensional lattice for exciting fluorescence, and uses a two-dimension small hole array 6 for filtering background fluorescence in the detecting light path, uses femtosecond pulsed laser with two different kinds of frequencies for composing a dual-color beam, wherein the two are coaxial, and the wavelengths are lambada 1 and lambada 2 respectively, moreover, lambada 1 is equal to lambada 2. Two kinds of light with different wavelength form two different three-dimensional lattice structures in the sample 3, and delay line 10 is modulated for causing the six beams to have the same optical path, only in the zone where two kinds of three-dimensional lattices coincide mutually, the dual-color two-photon fluorescence can be excitated. The device provided by the invention can effectively improve resolution response and imaging speed of three-dimensional imaging, and can be conveniently operated.

Description

technical field [0001] The present invention is related to microscopic imaging of biological tissues, and relates to two-photon fluorescence imaging, especially a two-color two-photon fluorescence imaging method and device capable of improving spatial resolution and imaging speed, so as to be applicable to the dynamic process of living biological cells in medicine detection. Background technique [0002] In recent years, two-photon excited fluorescence imaging has attracted widespread interest (Denk W, Strickler J, Webb W., Science, Vol. 248, 73-76, 1997). Two-photon excitation (hereinafter referred to as TPE) is a third-order nonlinear process. The characteristics of this nonlinear process make two-photon excitation require higher excitation power, and the intensity of the fluorescence signal is proportional to the square of the excitation light intensity. , so that the generation of two-photon fluorescence can be localized in the focal area. Therefore, two-photon imaging...

AI Technical Summary

Problems solved by technology

There are currently three methods to increase the imaging speed: the first is to use a high-speed scanner, such as a polygon mirror scanner (K.H.Kim, C.Buehler, P.T.C.So., Appl.Opt., Vol.38, 6004-6009, 1999), In this method, the increase in scanning speed will inevitably lead to a decrease in exposure time, thereby weakening the intensity of the fluorescent signal, which will reduce the signal-to-noise ratio (SNR)

The second is to scan only the limited volume of interest

These solutions can effectively increase the imaging speed and are very suitable for in vivo imaging, but their spatial resolution needs to be further improved

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