Structured illumination microscopy (SIM) is an established optical superresolution imaging technique. However, conventional SIM based on wide-field image acquisition is generally limited to visualizing thin cellular samples. We propose combining one-dimensional image rescan and structured illumination in the orthogonal direction to achieve superresolution without the need to rotate the illumination pattern. The image acquisition speed is consequently improved threefold, which is also beneficial for minimizing photobleaching and phototoxicity. Optical sectioning in thick biological tissue is enhanced by including a confocal slit in the system to significantly suppress the out-of-focus background and the associated noise. With all the technical improvements, our method captures three-dimensional superresolved image stacks of neuronal structures in mouse brain tissue samples for a depth range of more than 200 μm.
Laser speckle imaging has been an indispensable tool for in vivo imaging of blood flow in biological tissues. Here we report a novel design of laser speckle imaging system, which combines confocal illumination and detection with various speckle analysis methods. An illumination line is formed using a cylindrical lens and a 1-D scanning mirror is used to rapidly scan the line across the sample surface. The backscattered light is detected with a line camera at the confocal position. The acquired line speckle patterns can be analyzed with different methods, including temporal autocorrelation and spatial evaluation of speckle contrast, to retrieve the maps of correlation time and flow velocity. The line-scan configuration enables fast image acquisition, while confocal detection helps reject out-of-focus light and define a small focal volume. In vivo image experiments with chick embryos have demonstrated the excellent imaging performance, including depth selectivity, high spatial resolution for visualizing blood flow in the microvasculature, and high temporal resolution for dynamic flow quantification.
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