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Fast 3D imaging of giant unilamellar vesicles using reflected light‐sheet microscopy with single molecule sensitivity
Author(s) -
Jannasch Anita,
Szilagyi Sven A.,
Burmeister Moritz,
Davis Q. Tyrell,
Hermsdorf Gero L.,
De Suman,
Schäffer Erik
Publication year - 2022
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/jmi.13070
Subject(s) - photobleaching , light sheet fluorescence microscopy , microscopy , microscope , materials science , numerical aperture , optics , optical microscope , vesicle , optical sectioning , resolution (logic) , tracking (education) , membrane , scanning confocal electron microscopy , optoelectronics , chemistry , scanning electron microscope , fluorescence , physics , computer science , wavelength , biochemistry , psychology , pedagogy , artificial intelligence
Summary Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal‐to‐background ratio. To overcome these limitations, light‐sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light‐sheet microscope with active optics for fast, high contrast, two‐colour acquisition of z ‐stacks. We demonstrate fast volume scanning by imaging a two‐colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light‐sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single‐molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.