As high-resolution information is obtained only from the vicinity of the focal plane of the detection objective, with these methods the photon budget is increasingly wasted for increasingly thick samples. Unfortunately, many common methods of fluorescence microscopy (wide-field, structured illumination, 3D photoactivated localization, confocal and stimulated emission depletion microscopy) use an epi-illumination configuration that exposes the entire sample thickness to the illuminating radiation. ( c) To obtain images of acceptable SNR, a certain minimum number of photons must be emitted from these molecules, no matter how few they may be. ( b) Increasing temporal resolution demands that fewer molecules be expended (black spheres) at each time point. ( a) Increasing volumetric spatial resolution demands smaller voxels (boxes) encompassing fewer signal-generating fluorescent molecules (spheres). As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ~0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.Ĭhallenges in fluorescence imaging with high spatiotemporal resolution Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible.
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January 2023
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