Depth-dependent microscopic flow imaging with line scan laser speckle acquisition and analysis

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.