Single-cell proteomics is a challenging goal and an area of rapid methods development. This Focus issue highlights the many paths toward high-throughput, high-sensitivity measurements.

SODB is a large-scale platform providing rich data resources, efficient data exporting, and interactive analytical modules for spatial omics research.

Stimulated Raman scattering (SRS) microscopy has the capability to simultaneously visualize the spatial distribution of different biomolecules, but it remains challenging to reach super-resolution. To achieve this goal, a deconvolution algorithm, A-PoD, was developed and combined with SRS microscopy, enabling examination of nanoscopic biomolecular distribution and subcellular metabolic activity in cells and tissues.

Optimal design of spatial transcriptomic experiments allows statistical evaluation of the impact of various biological and technological features on the discovery of cell phenotypes.

ERnet is a deep learning-based software tool for automatic segmentation and classification of structures in the endoplasmic reticulum. ERnet is compatible with many fluorescence imaging modalities and can uncover subtle phenotypic changes.

Line-scan Brillouin microscopy enables fast 3D imaging of mechanical properties with low phototoxicity, as shown for Drosophila and mouse embryos, as well as ascidians.

A suite of tools including positive-going voltage indicators, a high-speed two-photon microscope, and denoising software enables prolonged imaging of electrical activity in neurons with limited toxicity.

Two mScarlet variants with high brightness and fast maturation times have been evolved. These variants behave favorably as fusion tags and Förster resonance energy transfer acceptors.

An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.

CNV-Y-DAMGO enables photopharmacological manipulations of mu opioid receptor signaling in behaving mice with high temporal resolution.

The nanopore community is stepping toward a new frontier of single-molecule protein sequencing. Here, we offer our opinions on the unique potential for this emerging technology, with a focus on single-cell proteomics, and some challenges that must be overcome to realize it.

The development of mass spectrometry-based single-cell proteomics technologies opens unique opportunities to understand the functional crosstalk between cells that drive tumor development.