Hermsdorf, G. L. | Search bioRxiv

bioRxiv2020

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

Observation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is 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 have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

biophysics↗

bioRxiv2020

Polycationic gold nanorods as multipurpose in vitro microtubule markers

Gold nanoparticles are intriguing because of their unique size- and shape-dependent chemical, electronic and optical properties. Various microscopy and biomedical applications are based on the particles biocompatibility, surface functionalizability, light absorption, and plasmon resonances. Gold nanorods (AuNRs) are particularly promising for various sensor applications due to their tip-enhanced plasmonic fields. For biomolecule attachment, AuNRs are often stabilized with amphiphilic molecules and functionalized with antibodies or biotin-binding proteins. However, by their intrinsic size such molecules block the most sensitive near-field region of the AuNRs. Here, we used short cationic thiols to covalently functionalize the gold surface. We show that the functionalization layer is thin and that these polycationic AuNRs bind in vitro to negatively charged microtubule filaments. Furthermore, we can plasmonically stimulate light emission from the AuNRs and, therefore, use them as bleach- and blinkfree microtubule markers. We confirmed colocalization by transmission electron microscopy or the combination of interference reflection and single-molecule fluorescence microscopy of fluorescently-labeled or plasmonic photoluminescent versions of the AuNRs. We expect that polycationic AuNRs may be applicable to in vivo systems and other negatively charged molecules like DNA. In the long-term, microtubule-bound AuNRs can be used as ultrasensitive single-molecule sensors for molecular machines that interact with microtubules.

biophysics↗