Light sheet (LS) illumination enables imaging of challenging biological samples by providing optical sectioning, thus reducing fluorescence background, photodamage, and photobleaching. Single-objective LS illumination, which uses the same objective lens for both illumination and detection, circumvents many of the optical complexities and constraints of conventional two- and multi-objective LS setups and simplifies sample mounting to a system that can be easily incorporated on a standard microscope stage. However, its implementation can be challenging due to the need for specialized imaging chambers that can precisely reflect the LS into the sample. Here, we present an adaptable and scalable approach for integrating reflective micromirrors into systems ranging from microfluidic channels to commercially available larger scale imaging chambers for single-objective LS microscopy. Our method employs 3D nanoprinting of micromirror structures that are metallized and coated for high reflectivity. The micromirror is then embedded into a customizable polydimethylsiloxane (PDMS) piece that acts as an insert for seamless incorporation into user-specified imaging chambers. The use of 3D nanoprinting, which is performed via two-photon polymerization, enables precise tuning of the micromirror dimensions and geometry to accommodate light sheets and samples of different dimensions. Furthermore, the use of PDMS as an insert, which is non-cytotoxic, nonfluorescent, and widely available, preserves chamber biocompatibility and functionality, as well as compatibility with other illumination modalities such as epi-fluorescence and transmission microscopy. Taken together, we think this approach will broaden access to single-objective LS microscopy and facilitate its integration into diverse biological applications.
Saliba et al. (Sun,) studied this question.