A high-resolution, easy-to-build light-sheet microscope for subcellular imaging.

Abstract

Although several open-source, easy-to-assemble light-sheet microscope platforms already exist-such as mesoSPIM, OpenSPIM, and OpenSpin-they are optimized for imaging large specimens and lack the resolution required to visualize subcellular features, such as organelles or cytoskeletal architectures. In contrast, lattice light-sheet microscopy (LLSM) achieves the resolution necessary to resolve such fine structures but, in its open-source implementation, can be alignment- and maintenance-intensive, often requiring specialist expertise. To address this gap, we developed Altair light-sheet fluorescence microscopy (LSFM), a high-resolution, open-source, sample-scanning light-sheet microscope specifically designed for subcellular imaging. By optimizing the optical pathway in silico, we created a custom baseplate that greatly simplifies alignment and assembly. The system integrates streamlined optoelectronics and optomechanics with seamless operation through our open-source software, <i>navigate</i>. Altair-LSFM achieves lateral and axial resolutions of approximately 235 and 350 nm, respectively, across a 266 µm field of view after deconvolution. We validate the system's capabilities by imaging sub-diffraction fluorescent nanospheres and visualizing fine structural details in mammalian cells, including microtubules, actin filaments, nuclei, and Golgi apparatus. We further demonstrate its live-cell imaging capabilities by visualizing microtubules and vimentin intermediate filaments in actively migrating cells.