Abstract
Super-resolution imaging and single-particle tracking require cells to be immobile as any movement reduces the resolution of the measurements. Here, we present a method based on APTES-glutaraldehyde coating of glass surfaces to immobilize cells without compromising their growth. Our method of immobilization is compatible with Saccharomyces cerevisiae, Escherichia coli, and synthetic cells (here, giant-unilamellar vesicles). The method introduces minimal background fluorescence and is suitable for imaging of single particles at high resolution. With S. cerevisiae we benchmarked the method against the commonly used concanavalin A approach. We show by total internal reflection fluorescence microscopy that modifying surfaces with ConA introduces artifacts close to the glass surface, which are not present when immobilizing with the APTES-glutaraldehyde method. We demonstrate validity of the method by measuring the diffusion of membrane proteins in yeast with single-particle tracking and of lipids in giant-unilamellar vesicles with fluorescence recovery after photobleaching. Importantly, the physical properties and shape of the fragile GUVs are not affected upon binding to APTES-glutaraldehyde coated glass. The APTES-glutaraldehyde is a generic method of immobilization that should work with any cell or synthetic system that has primary amines on the surface.
Highlights
Fluorescence microscopy is a common method for studies of biological processes
The modification of the glass cover slides by APTES w/wo glutaraldehyde was investigated with contact angle measurements; details of the method are described in the Methods section under “Preparation and characterization of coverslips”
The here described APTES-glutaraldehyde method proves versatile for different types of cells including synthetic lipid vesicles and gives reduced background fluorescence when compared with concanavalin A (ConA)
Summary
Fluorescence microscopy is a common method for studies of biological processes. Information about the localization, interactions and structure of macromolecules and sub-cellular organization of the cell can be obtained. The trajectories of individual molecules are traced for a long period of time Both high-resolution imaging and tracking of fluorescent molecules require cells to be relatively immobile, as any movement of the cell will decrease the localization accuracy of the fluorophore. In single-particle tracking or super-resolution imaging experiments the fluorophores can be localized with an accuracy of 20–50 nm. A method based on electrostatic interactions does not work for all cells, including Saccharomyces cerevisiae In those cases, the carbohydrate-binding protein concanavalin A (ConA)[15], a lectin, has been used for immobilization. We developed a generic method to immobilize cells for super-resolution imaging and single particle tracking measurements based on APTES coating of glass slides, followed by glutaraldehyde treatment and subsequent reaction with amines on the surface of the cells (Fig. 1a). The cells can be imaged in any solution and grow in the proper media during the imaging
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