Hard-wired lattice light-sheet microscopy for imaging of expanded samples.

Light-sheet fluorescence microscopy (LSFM) helps investigate small structures in developing cells and tissue for three-dimensional localization microscopy and large-field brain imaging in neuroscience. Lattice light-sheet microscopy is a recent development with great potential to improve axial resolution and usable field sizes, thus improving imaging speed. In contrast to the commonly employed Gaussian beams for light-sheet generation in conventional LSFM, in lattice light-sheet microscopy an array of low diverging Bessel beams with a suppressed side lobe structure is used. We developed a facile elementary lattice light-sheet microscope using a micro-fabricated fixed ring mask for lattice light-sheet generation. In our setup, optical hardware elements enable a stable and simple illumination path without the need for spatial light modulators. This setup, in combination with long-working distance objectives and the possibility for simultaneous dual-color imaging, provides optimal conditions for imaging extended optically cleared tissue samples. We here present experimental data of fluorescently stained neurons and neurites from mouse hippocampus following tissue expansion and demonstrate the high homogeneous resolution throughout the entire imaged volume. Utilizing our purpose-built lattice light-sheet microscope, we reached a homogeneous excitation and an axial resolution of 1.2 µm over a field of view of (333 µm)2.

Light-sheet fluorescence expansion microscopy: fast mapping of neural circuits at super resolution.

The goal of understanding the architecture of neural circuits at the synapse level with a brain-wide perspective has powered the interest in high-speed and large field-of-view volumetric imaging at subcellular resolution. Here, we developed a method combining tissue expansion and light-sheet fluorescence microscopy to allow extended volumetric super resolution high-speed imaging of large mouse brain samples. We demonstrate the capabilities of this method by performing two color fast volumetric super resolution imaging of mouse CA1 and dentate gyrus molecular-, granule cell-, and polymorphic layers. Our method enables an exact evaluation of granule cell and neurite morphology within the context of large cell ensembles spanning several orders of magnitude in resolution. We found that imaging a brain region of 1 mm 3 in super resolution using light-sheet fluorescence expansion microscopy is about 17-fold faster than imaging the same region by a current state-of-the-art high-resolution confocal laser scanning microscope.