# Volumetric Single‐Molecule Tracking Inside Subcellular Structures

## TL;DR

This paper introduces a new microscopy method to track molecules in 3D within cells, revealing how disease-related proteins affect molecular movement in organelles.

## Contribution

A correlative microscopy approach combining SMLFM and FLFM for 3D single-molecule tracking in living mammalian cells.

## Key findings

- Instantaneous volumetric segmentation improves sensitivity in measuring molecular organization and diffusion.
- Heterogeneous molecular motion was observed in endoplasmic reticulum inclusions linked to α1-antitrypsin deficiency.

## 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 α1‐antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.

This correlative microscopy approach integrates single‐molecule light‐field microscopy (SMLFM) with widefield Fourier light‐field microscopy (FLFM) to enable 3D single‐molecule tracking within the organelles of living mammalian cells. Instantaneous volumetric segmentation enhances the sensitivity of molecular organization and diffusion measurements, revealing heterogeneous molecular motion within the endoplasmic reticulum of cells expressing disease‐associated α1‐antitrypsin polymers.