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bioRxiv · 10.1101/2025.10.23.684144

STED-FCS in subdiffraction limit volumes reveals altered diffusion in live cell applications

Zobiak, B.·Andres-Alonso, M.·Wang, Y.·Mohammadi, B.·Gorzelanny, C.·Altmeppen, H. C.·Yin, S.·Failla, A. V.
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Abstract

Fluorescence correlation spectroscopy (FCS) is a widely established light microscopy technique for investigating physiological parameters such as diffusion states, particle numbers, and viscosity in biological samples. Combining FCS with stimulated emission depletion (STED-FCS) has enabled the investigation of molecular diffusion to sub diffraction-limited volumes. However, the full potential of STED-FCS for biomedical applications remains underexplored. Here, we present proof-of-principle studies with novel implementations of STED-FCS for investigating biological processes in living samples. Specifically, we demonstrate the impact of STED-FCS analyses in three distinct biomedical assays. Firstly, we prove that STED-FCS is capable of observing dynamic changes in autophagy-related protein microtubule-associated protein 1 light chain 3b (LC3b) along neuronal axons. Secondly, we show that STED-FCS can resolve alterations in the carbohydrate chain lengths of glycoproteins in melanoma cells. Finally, we demonstrate that STED-FCS can measure the reduced mobility of lipids within the plasma membrane of neuronal cells treated with the Alzheimers disease-associated, aggregation-prone toxic peptide amyloid-beta 1-42 (A{beta}42). We believe that this study will inspire researchers to utilize STED-FCS to address critical questions in their bio-imaging studies, particularly regarding the super-resolution assessment of dynamic processes in living cells.

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Zobiak, B., Andres-Alonso, M., Wang, Y., Mohammadi, B., Gorzelanny, C., Altmeppen, H. C., Yin, S., Failla, A. V.. 2025-10-24. STED-FCS in subdiffraction limit volumes reveals altered diffusion in live cell applications. https://doi.org/10.1101/2025.10.23.684144

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