Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells

Le résumé fourni par la source

An ideal tool for the study of cellular biology would enable the measure of molecular activity nondestructively within living cells. Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells but have historically been limited by a necessary tradeoff between spatiotemporal resolution and throughput. Here we address these limitations using oblique line scan (OLS), a robust single-objective light-sheet-based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a large field of view. We show that OLS can be used to capture protein motion up to 14 μm2 s−1 in living cells. We further extend the utility of OLS with in-solution SMT for single-molecule measurement of ligand–protein interactions and disruption of protein–protein interactions using purified proteins. We illustrate the versatility of OLS by showcasing two-color SMT, STORM and single-molecule fluorescence recovery after photobleaching. OLS paves the way for robust, high-throughput, single-molecule investigations of protein function required for basic research, drug screening and systems biology studies. Oblique line scan microscopy achieves nanoscale spatial and sub-millisecond temporal resolution across a large field of view, enabling improved and robust single-molecule biophysical measurements and single-molecule tracking in both cells and solution.

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