Research On The Random Access Two-photon Fluorescence Microscope

Two-photon fluorescence microscopy (TPM) has now grown up to be an important technique in biology especially neuroscience research. However, as a kind of scanning microscopy, the temporal resolution of TPM cannot easily satisfy the requirement for the detection of fast functional signal. The temporal resolution is improved by using the random access scan method and scanning only those regions of interest. A random access two-photon fluorescence microscope is constructed. The study of the main problem in the construction of the system is discussed in this dissertation.The method of using the random access ability of the acousto-optic deflector (AOD) for increasing the acquisition rate of the signal from the regions of interest is discussed. By restricting the scan position to a few regions of interest and utilizing the fast positioning ability of the AOD, more time can be used for fluorescence signal integration and average and the effective temporal resolution can be increased while a certain signal to noise ratio still remains.A new single prism dispersion compensation scheme is proposed to compensate for the dispersion effect of the AODs on the ultrashort-pulsed excitation laser. A single prism is introduced to compensate for the spatial dispersion effect of the AODs and the AODs are also used as one part of the temporal dispersion compensation unit. The temporal dispersion due to the material dispersion of the AOD is compensated by the temporal dispersion effect of the angular dispersion of the prism-AOD structure. Effective software calibration technique by discarding the initial data set is used to solve the problem caused by the delay between scan positioning and scan control signal.The major problems in using LabVIEW for the development of the system software are discussed, and a well structured LabVIEW program framework, which can be easily implemented and maintained, is designed.The constructed system is applied for calcium imaging in the brain slice. The relationship between high frequency firing of action potential and the calcium fluorescence signal of the neuron is verified. The noise of the system is analyzed and the shot noise is believed to be the main noise source at the brain slice experiment signal level.Using a 60X oil immersion objective (N.A. = 1.42), 0.3μm lateral resolution and 1.3μm axial resolution can be obtained for the random access system when the N.A. of the objective is fully utilized. Under the same excitation power level, with dispersion compensation, the fluorescence intensity is 15 times as high as the case without dispersion compensation. 10μs per scan point scan speed can be realized, which is 100 times as fast as the imaging speed using galvanometer driven scanning mirror. The random access system is capable of, in a single trial, recording the variation of the calcium fluorescence signal from the region of interest corresponding to the firing of action potential at 50 Hz with single action potential resolution.