![]() In contrast, thicker Gaussian light sheets can image larger areas, but have lower resolution and optical sectioning. Thinner Gaussian light sheets provide greater axial resolution and optical sectioning, but at the cost of a shorter propagation length and smaller field of view. ![]() ![]() This approach results in an inherent tradeoff between the thickness of the light sheet and its propagation length. The most common implementations of light sheet microscopy use cylindrical lenses to focus a Gaussian beam into a laterally extended sheet with a Gaussian axial intensity profile at the specimen. Although point-scanning methods like confocal also increase axial resolution and optical sectioning, plane illumination with widefield detection enables 100 to 1000 times faster imaging speed with dramatically lower photobleaching 5. Thus, if the illumination pattern is comparable to or thinner than the detection depth of field, single plane illumination can also enhance axial resolution. Moreover, in fluorescence microscopy, the overall point spread function (PSF) is a product of both the excitation and detection PSFs. This minimizes out-of-focus illumination, reduces photobleaching, and increases signal-to-noise ratio (SNR) compared to epifluorescence and confocal microscopy. Light sheet microscopy only illuminates a thin plane at the specimen. Over the last two decades, light sheet microscopy has been used to image biological samples of various scales, ranging from single molecules to whole organisms 1, 2, 3, 4. Finally, we introduce an approach to spectrally fuse sequential acquisitions of different lattice light sheet patterns with complementary optical properties to achieve both high resolution and low background images. We demonstrate how different optical lattice illumination patterns can be tuned to prioritize either axial resolution or optical sectioning. Using simulations and experimental measurements in fixed and live cells, we quantify the differences between Gaussian and lattice light sheets on beam uniformity, axial resolution, lateral resolution, and photobleaching. However, these advantages come at the expense of an increased total illumination to the specimen and a decreased axial confinement of the illumination pattern. Light sheets based on dithered optical lattices improve axial resolution and beam uniformity compared Gaussian beams by using axially structured illumination patterns. However, when equipped with Gaussian beams, the axial resolving power of a light sheet microscope and the observable field of view are inversely related. Light sheet microscopes reduce phototoxicity and background and improve imaging speed compared to widefield and confocal microscopes.
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