Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects.

Adaptive optics (AO) assisted microscopy enables high-quality imaging by correcting instrument- and specimen-induced aberrations, utilizing wavefront shaping devices such as a deformable mirror (DM). For optimal wavefront modulation, it is crucial to position the DM accurately at the conjugate pupil plane and align it symmetrically with the optical axis. However, achieving these requirements can be challenging for researchers lacking a background in AO instrumentation. Here, we propose a simple and practical approach for precisely adjusting the three-dimensional position of the DM using a custom-made bead sample. The experimental results demonstrate the effectiveness of this approach, achieving uniform phase modulation across a total field of view of 60 μm × 60 μm, with a standard deviation of <0.1 λ/2π in the decomposed Zernike coefficients from Z5 to Z25 in Wyant order.

Biplane single-molecule localization microscopy (SMLM) enables three-dimensional (3D) super-resolution imaging by extracting the axial position of fluorophores from a pair of emission patterns detected at two axially separated planes. The separation between these two imaging planes, termed the biplane distance, is a key parameter that determines axial localization precision, yet a systematic investigation of its optimal selection remains lacking. Here, we calculate the theoretical localization precision across a range of biplane distances and identify an optimal value, construct a tunable biplane detection module and experimentally evaluate axial localization precision at three different biplane distances using both fluorescent beads and biological specimens. Experimental results confirm the theoretical predictions and provide a practical framework for optimizing the biplane distance in 3D-SMLM systems.