| Background rejection is a standard method of improving image contrast and conferring 3D resolution in fluorescence microscopy. However, imaging techniques which offer background rejection often require a scanning illumination system which typically limits imaging speed. We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. This hybrid illumination technique requires the acquisition of two images, one standard image with uniform illumination, and a second image with speckle illumination. From a priori knowledge of speckle statistics, an examination of imaged speckle contrast in the speckle image provides an optically-sectioned, albeit low-resolution image. This low-resolution image can then be fused with high-resolution details extracted from the uniform image in order to recover an optically-sectioned image with full (diffraction-limited) lateral resolution. For this reason, we refer to this technique as HiLo microscopy.;The most ubiquitous tool for optically sectioned microscopy is the confocal laser scanning microscope. A direct comparison shows that HiLo microscopy provides imaging performance comparable to that of a state-of-the-art confocal system even in thick tissue. Besides the fast imaging speed inherent to wide-field acquisition, HiLo microscopy provides a tunable depth of field that can be set a posteriori by a single image processing parameter. We show that HiLo microscopy is robust, versatile, simple to implement and is well-suited for in vivo imaging. |
