Versatile and Scalable Reflective Micromirrors for Single-Objective Light Sheet Microscopy

We present a tunable microfabrication pipeline for creating robust, reflective inserts that adapt conventional commercial imaging chambers for single-objective light sheet (LS) illumination. This system reduces the complexity associated with dual-objective LS setups and specialized LS chambers while retaining the native functionality and biocompatibility of the original chambers. The fabricated insert features a metalized, 3D nanoprinted micromirror with an angled reflective surface, enabling alignment of a thin LS for sectioning and imaging throughout mammalian cells. Using this pipeline, we demonstrate that single-objective LS illumination achieves an over 4X improvement in the signal-to-background ratio compared with conventional widefield epiillumination in both fixed and live cell samples. Furthermore, we show substantial resolution enhancement for single-molecule localization microscopy compared to epi-illumination for improved imaging at the nanoscale. The versatile and scalable design offers an easily implemented approach to bring the benefits of single-objective LS microscopy to a wide array of biological studies.