Abstract
Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal resolution. However, the creation of thin light-sheets for high axial resolution is challenging, as the thickness of the sheet, field of view and confinement of the excitation need to be carefully balanced. Some of the thinnest light-sheets created so far have found little practical use as they excite too much out-of-focus fluorescence. In contrast, the most commonly used light-sheet for subcellular imaging, the square lattice, has excellent excitation confinement at the cost of lower axial resolving power. Here we leverage the recently discovered Field Synthesis theorem to create light-sheets where thickness and illumination confinement can be continuously tuned. Explicitly, we scan a line beam across a portion of an annulus mask on the back focal plane of the illumination objective, which we call C-light-sheets. We experimentally characterize these light-sheets and demonstrate their application on biological samples.
Highlights
Light-sheet fluorescence microscopy (LSFM) has been transformative for volumetric imaging of single cells up to entire organisms, as it minimizes sample irradiation and allows efficient and rapid 3D imaging[1,2,3]
When propagation invariant beams are used for DSLM, for a given light-sheet thickness, any increase in confocal parameter is paid by reduced confinement of the excitation energy into the sheet
If we look at the corresponding optical transfer function (OTF), one can see that the support is elongated in the axial direction, the strength further away from the center is weak
Summary
Light-sheet fluorescence microscopy (LSFM) has been transformative for volumetric imaging of single cells up to entire organisms, as it minimizes sample irradiation and allows efficient and rapid 3D imaging[1,2,3]. Some LSFM implementations have been developed that can attain 300nm scale axial resolution[3,4,5,6,7], effectively doubling the resolution of confocal microscopy, the workhorse in 3D microscopy This is enabled by the larger set of angles that the separate illumination and detection objectives cover compared to a single objective microscope system. Sheets with sub-micron thickness can be created, their confocal parameter is usually shorter than a typical cell, which makes them ill-suited for practical imaging Propagation invariant beams, such as Bessel[8] and Airy[9, 10] beams, and optical lattices, can in principle overcome the divergence of Gaussian beams. Large amounts of out-of-focus light are excited, which generate unwanted out-of-focus fluorescence and lead to accelerated photo-bleaching
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