| Summary: | Fluorescence microscopy provides an unparalleled tool for imaging biological samples. However, producing high-quality volumetric images quickly and without excessive complexity remains a challenge. Here, we demonstrate a four-camera structured illumination microscope (SIM) capable of simultaneously imaging multiple focal planes, allowing for the capture of 3D fluorescent images without any axial movement of the sample. This setup allows for the acquisition of many different 3D imaging modes, including 3D time lapses, high-axial-resolution 3D images, and large 3D mosaics. We imaged mitochondrial motions in live cells, neuronal structure in <i>Drosophila</i> larvae, and imaged up to 130 µm deep into mouse brain tissue. After SIM processing, the resolution measured using one of the four cameras improved from 357 nm to 253 nm when using a 30×/1.05 NA objective.
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