Volumetric Single‐Molecule Tracking Inside Subcellular Structures

ABSTRACT The non‐covalent interactions that underpin major cellular functions depend on molecular motion within 3D environments. Large depth‐of‐field single‐molecule localization microscopy (3D‐SMLM) methods facilitate these measurements, but their increased optical complexity and bespoke post‐processing pipelines often sacrifice important cellular context. Here, we combine single‐molecule light‐field microscopy (SMLFM) with widefield Fourier light‐field microscopy for correlative volumetric organelle imaging. The instantaneous acquisition of subcellular volumes improves the sensitivity of molecular organization, chemical environment, and diffusion measurements through the use of volumetric sub‐cellular segmentation. We first demonstrate our approach by measuring the molecular organization of a nuclear‐localized HaloTag protein relative to cell nuclei. Next, we characterize the molecular diffusion of the soluble protein, calreticulin, in the context of ‐antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.