Research on the Random Access Two-photon Fluorescence Microscope
|School||Huazhong University of Science and Technology|
|Keywords||Two-photon fluorescence microscopy imaging Acousto-optic deflector Dispersion compensation Random scan Calcium imaging|
Two-photon fluorescence microscopy imaging technology has gradually become the life sciences, particularly neuroscience technology. However, two-photon fluorescence microscopy imaging is a scanning imaging technology, the time resolution is not high, can not satisfy the functional biology research for the rapid changes in the function of signal detection needs. The time resolution of only random scan mode to scan the region of interest to solve the two-photon fluorescence microscopy imaging techniques to achieve random scanning two-photon fluorescence microscopy imaging system. In this paper, the study of the main problems in the system are discussed. The use of acousto-optic deflector random scanning the ability to improve the resolution of the method of the region of interest signal acquisition time. By scanning position limits in certain regions of interest, high-speed scanning using acousto-optic deflector positioning capability to shorten the scan time, more time for the accumulation and the average of the fluorescence signal, thereby enhancing the effective time resolution, while ensuring a certain signal-to-noise ratio. New single prism dispersion compensation method to solve the acousto-optic deflector ultrashort pulse excitation light dispersion. The introduction of a single prism compensation acousto-optic deflector spatial dispersion, and acousto-optic deflector at the same time as an integral part of the temporal dispersion compensation unit prism acousto-optic deflector composed of dispersion compensation structure angular dispersion introduced sound and light deflector material dispersion introduced temporal dispersion. Using discarded the initial data segment, the problem of the acousto-optic deflector scan positioning and scanning control signal delay between the signal dislocation problem. The LabVIEW program framework LabVIEW for the rapid development of the system software, designed to facilitate the implementation and maintenance. Random scanning system is applied to calcium fluorescence imaging of the brain slices to verify the correspondence between the high frequency of neurons, spiking and calcium fluorescence signal. In the practical application of the system noise sources pointed out that the fluorescence signal level in the biological experiments, shot noise is the main source of noise. Random scanning two-photon fluorescence microscopy imaging system designed and implemented in the full use of the numerical aperture of an objective lens for 60X oil immersion lens (numerical aperture of 1.42), can reach the vertical resolution of the lateral resolution of 0.3μm and 1.3μm. And spent fluorescent signal intensity after the dispersion compensation in the case of the sample at the same power, is about 15 times before dispersion compensation. Maximum scanning speed of per scan 10 microseconds, is commonly used in scanning mirror scan imaging speed is about 100 times, can be observed with a single action potential resolution to the neurons in the region of interest corresponding to 50 Hz, spiking calcium fluorescence signal changes.