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

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 Alzheimer’s disease-associated, aggregation-prone toxic peptide amyloid-beta 1-42 (Aβ42). 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.