Single-molecule localization microscopy (SMLM) enables imaging beyond the diffraction limit, facilitating the investigation of cellular structures and mechanisms at the nanoscale. Light sheet illumination enhances SMLM by reducing the fluorescent background, which leads to improved signal-to-background ratio and localization precision. Lattice light sheet (LLS) microscopy, which employs a 2D optical lattice for light sheet illumination, has been demonstrated to offer effective imaging performance across a variety of biological samples. However, its conventional dual-objective design poses challenges for certain applications in imaging of mammalian cells. Here, we present a new imaging platform which implements LLS illumination with a reflective single-objective geometry (soLLS) inside a microfluidic chip, enabling the use of a single high numerical aperture objective for both illumination and detection, mitigating constraints of the convention LS configuration and facilitating solution exchange with microfluidics. In this work, we provide a quantitative characterization of the propagation properties of the soLLS and demonstrate its advantages over conventional illumination strategies in terms of sectioning, effective sectioning range, penetration depth in scattering samples, and imaging performances in mammalian cell samples. By combining soLLS with point spread function engineering in the emission path, we demonstrate the platform for improved 3D single-molecule super-resolution imaging of multiple targets across multiple cells using Exchange-PAINT. The soLLS imaging platform offers the potential to visualize nanoscale cellular and intercellular structures in challenging samples, thereby expanding and enhancing a wide range of applications in biology and biomedicine.
Cheng et al. (Sun,) studied this question.