The Study Of Axial Random-Access Laser Scanning Scheme In Two Photon Microscopy

Two photon microscopy has played an important role in the study of neuroscience due to its advantage of deep imaging, low damage to biological sample, etc. The recently developed random-access two photon microscopy, which is able to selectedly scan any separated point in the field of view at very high speed, has shown its advantage in the study of neuronal function activity. Furthermore, benefiting from the development of the theory that describes dispersion characteristics of femtosecond laser after passing through angular elements, random-access two photon microscope becomes more practical. Neuronal structure spans in three-dimesions, accordingly the fast fluorescence signal that is related to neuronal functional activity is distributed in three-dimensions. Thus, it is necessary to develop three-dimensional random-access two photon microscopy to detect such signals. To this end, the key challege is to develop fast axial random-access laser scanning method, because of the achievement of random-access laser scanning in two dimensions (x-y plane). The conventional axial laser scanning is realized by mechanically moving the objective or the sample in axial direction, inducing slow scanning speed limited by mechanical intertia. It is possible to fast axially move laser focus in two photon microscope by changing the divergence of input beam, however, pulse-broadening, low axial scanning range, etc. occurs when applying current axial scanning scheme in two photon microscope.To develop high efficiency, practical fast three-dimensional random-access microscope, it is necessary to study dispersion compensation method and axial scanning mechanism. Here in this dissertation, novel dispersion compensation scheme and axial scanning methods are studied. The main innovative results are:(1) The relationship between laser beam size and dispersion compensation capability of angular elements is revealed. It is found that, with expanded beam, the maximum negative group delay dispersion provided by angular elememts (take prism for example) increases by an order of magnitude compared with original beam; when using prisms or acousto-optical deflectors to compensate positive group delay dispersion with a modest 2×and 4×expanded beams, the restored minimal pulse width decreases by 50% and the corresponding distance between angular elements is shortened more than 70 cm compared with original beam.(2) The fast axial laser scanning scheme has been developed by the combination of fast dispersion manipulation capability of acousto-optic deflector and temporal focusing technique. The relationship between the axial shift of temporal focus and acoustic frequency in acousto-optic deflector is revealed. The shift range of temporal focus is 9μm using the acousto-optic deflectors operated from 80 MHz to 120 MHz. The speed of position change of temporal foucs is of the order 10μm, which is dependent on the transit time of acousto-optic deflector.(3) The axial random-access laser scanning scheme based on device of KTN crystal is studied. The relationship between design parameters and the focal length of KTN lens is revealed; the dependence of axial scanning range on desige parameter of KTN lens and applied voltage is revealed.