A fluorescent emission HIST microscope utilizes a highly inclined tile beam that is scanned over a biological sample object. Fluorescence emission from the sample is propagated through a confocal slit into an sCMOS camera supporting a rolling shutter mode. The tile beam is synchronously swept with the readout of the camera to facilitate the rejection of background. The system provides for decoupling of the total imaging area from the beam thickness, which now solely depends on the width of the tile beam, enabling a thinner illumination and larger FOV imaging. Clear visualization of single molecules across a 130 μm×130 μm FOV provided a greater than 40× FOV than conventional HILO imaging. An associated imaging method is disclosed.
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