The topology of the cerebral vasculature, which is the energy transport corridor of the brain, can be used to study cerebral circulatory pathways. Limited by the restrictions of the vascular markers and imaging methods, studies on cerebral vascular structure now mainly focus on either observation of the macro vessels in a whole brain or imaging of the micro vessels in a small region. Simultaneous vascular studies of arteries, veins and capillaries have not been achieved in the whole brain of mammals. Here, we have combined the improved gelatin-Indian ink vessel perfusion process with Micro-Optical Sectioning Tomography for imaging the vessel network of an entire mouse brain. With 17 days of work, an integral dataset for the entire cerebral vessels was acquired. The voxel resolution is 0.35×0.4×2.0 µm3 for the whole brain. Besides the observations of fine and complex vascular networks in the reconstructed slices and entire brain views, a representative continuous vascular tracking has been demonstrated in the deep thalamus. This study provided an effective method for studying the entire macro and micro vascular networks of mouse brain simultaneously.

Femtosecond laser pulses have made a revolution in multiphoton excitation microscopy, micromachining, and optical storage for their unprecedented high peak power. However, modulation of their intensity with acousto-optic modulator (AOM) is frustrated by dispersion which results in a significant stretch in pulse width. Here we report a scheme composed of two acousto-optic deflectors (AODs) to modulate the intensity of the femtosecond laser pulses with simultaneous compensation for the temporal dispersion. With commercial AODs, we demonstrated such an AOM system for the femtosecond laser pulses with overall transmission efficiency of around 80%. The pulse width of the exit beam is 115-177 fs for an input pulse of 110 fs, across the wavelength range of 720-920 nm when the temporal dispersion compensation is optimally tuned at 800 nm. The fluorescence intensity in a two-photon microscopy experiment performed using this system increased 5.5-fold over that of the uncompensated AOM.

2009) Analysis of fast axial scanning scheme using temporal focusing with acousto-optic deflectors, Journal of Modern Optics, 56:1, 81-84, Multiphoton microscopy has gained broad application in biology and medicine in the recent decade by assessing biological structure and function with high spatial resolution. Recently it has achieved fast two-dimensional x-y imaging by using inertia-free scanning mechanisms. However, the axial scanning speed is severely limited by mechanical inertia. This paper proposes a fast axial scanning scheme using temporal focusing implemented with an inertia-free scanning device, such as an acousto-optic device (AOD), and presents an experimental demonstration of two-photon axial scanning over a 9 mm range using AODs operated from 80 to 120 MHz. A theoretical analysis provides the detailed characteristics of this method. The effects of both the ratio of collimating lens to objective lens focal lengths or magnification (M), and the degree of group delay dispersion (GDD) in the light source on the temporal focusing and axial scanning range are described by theory and experiments.

The performance of a dispersion-compensated acousto-optic deflector (AOD) for steering femtosecond laser pulses was examined with the prism located before or after the AOD, which is regarded as prism-AOD and AOD-prism, respectively. Comparisons are made over parameters including the spot spatial pattern, output pulse width, scanning linearity, the field of view, and the transmission rate. Fluorescence images of 170 nm diameter beads and cells were measured to provide an overall evaluation for these femtosecond laser beam scanning configurations. On the basis of these experiments, the prism-AOD configuration is concluded to be more advantageous for the purpose of simultaneous compensation for the spatial and temporal dispersion.