Abstract
Quantifying dynamics of complex macromolecules in cellula or in vivo has been particularly challenging owing to barriers in instrumentation, fluorescence methodologies and tracking freely diffusing molecules within living cell. Here we take a bottom-up engineering approach to successfully solve these barriers by coupling single-molecule FRET (smFRET), which helps to quantify dynamics at nanoscales, with in vitro site-specifically labelled proteins (Hsp90)introduced into mammalian cells and zebrafish (Danio rerio) embryos via a live-cell injector.Hsp90 machinery with its two distinct major conformational states provides a model representative macromolecule to re-examine conformational changes in cellula and in vivo.The injected proteins are visualized in a custom-built live-cell smFRET Highly Inclined Laminar Optical Light sheet (HILO) microscope.
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