Scanned light sheet microscopy with confocal slit detection

Abstract

In light sheet fluorescence microscopy optical sectioning is achieved by illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet fluorescence excitation and confocal slit detection. A high frame rate was achieved by using the "rolling shutter" of a scientific CMOS camera as a slit detector. Synchronizing the "rolling shutter" with the scanned illumination beam results in confocal line detection. Acquiring image data with selective plane illumination minimizes photo-damage while simultaneously enhancing contrast, optical sectioning and signal-to-noise ratio. Thus the imaging principle presented here merges the benefits of scanned light sheet microscopy and line-scanning confocal imaging.