Axially overlapped multi-focus light sheet with enlarged field of view

Abstract

Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multifocus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish.

Interferometric modulation for generating extended light sheet: improving field of view

“…However, these methods require additional camera exposure time, sacrificing imaging speed. In recent years, the concept of multiple LS illuminations has been proposed to expand the FOV in LSFM without sacrificing camera exposure time, 27 , 28 thereby maintaining LSFM imaging speed. However, due to inevitable mutual interference between multiple LS, researchers have implemented measures such as laterally separating the beams and subtracting complementary images 28 or precisely designing the optical path length of each beam in the optical system to exceed the coherence length of the light source 27 .…”

“…However, due to inevitable mutual interference between multiple LS, researchers have implemented measures such as laterally separating the beams and subtracting complementary images 28 or precisely designing the optical path length of each beam in the optical system to exceed the coherence length of the light source. 27 These measures are taken to avoid interference between LSs. However, it is important to note that these additional measures increase the complexity of the system.…”

Laterally swept light-sheet microscopy enhanced by pixel reassignment for photon-efficient volumetric imaging

“…12 Another method is to use multiple light sheets with swept focus 13 or tiled beams. [14][15][16][17] Swept focus light-sheet microscopy (SFLM) generates a virtual light sheet with a higher aspect ratio. However, multiple beams also introduce more sidelobes, which decrease the axial resolution and worsen the optical sectioning.…”

Motionless synthesis and scanning of lattice light sheets with a single digital micromirror device

“…That is, a lattice light sheet is a super-resolution light sheet. Thus, compared to conventional lightsheet microscopy, [10][11][12][13][14] lattice light-sheet microscopy simultaneously optimizes the axial resolution (decided by the thickness of the light sheet) and field of view (FOV, the length over which the light sheet can persist its shape) and further increases the speed of image acquisition and reduces phototoxicity.…”