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bioRxiv · 10.1101/2025.06.09.658280

Volumetric single-molecule tracking inside subcellular structures

Daly, S.·Chambers, J. E.·Jones, C.·Fu, B.·Manton, J. D.·Beckwith, J. S.·Marciniak, S. J.·Gershlick, D. C.·Lee, S. F.
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Abstract

The molecular interactions that underpin all cellular functions depend on molecular motion within three-dimensional 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 can 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 and diffusion measurements through the rejection of non-specific localizations. 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 1-antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.

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Daly, S., Chambers, J. E., Jones, C., Fu, B., Manton, J. D., Beckwith, J. S., Marciniak, S. J., Gershlick, D. C., Lee, S. F.. 2025-06-10. Volumetric single-molecule tracking inside subcellular structures. https://doi.org/10.1101/2025.06.09.658280

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