Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking

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...

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Autores principales:	Łukasz Syga, Dian Spakman, Christiaan M. Punter, Bert Poolman
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Single Particle Tracking (SPT) Total Internal Reflection Fluorescence (TIRF) Fluorescence Recovery After Photobleaching (FRAP) Super-resolution Imaging Immobilized Cells Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ad83b3b67e6f4a94848e1347944bf434
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spelling	oai:doaj.org-article:ad83b3b67e6f4a94848e1347944bf4342021-12-02T15:08:26ZMethod for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking10.1038/s41598-018-32166-y2045-2322https://doaj.org/article/ad83b3b67e6f4a94848e1347944bf4342018-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-32166-yhttps://doaj.org/toc/2045-2322Abstract 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.Łukasz SygaDian SpakmanChristiaan M. PunterBert PoolmanNature PortfolioarticleSingle Particle Tracking (SPT)Total Internal Reflection Fluorescence (TIRF)Fluorescence Recovery After Photobleaching (FRAP)Super-resolution ImagingImmobilized CellsMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-12 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Single Particle Tracking (SPT) Total Internal Reflection Fluorescence (TIRF) Fluorescence Recovery After Photobleaching (FRAP) Super-resolution Imaging Immobilized Cells Medicine R Science Q
spellingShingle	Single Particle Tracking (SPT) Total Internal Reflection Fluorescence (TIRF) Fluorescence Recovery After Photobleaching (FRAP) Super-resolution Imaging Immobilized Cells Medicine R Science Q Łukasz Syga Dian Spakman Christiaan M. Punter Bert Poolman Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
description	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.
format	article
author	Łukasz Syga Dian Spakman Christiaan M. Punter Bert Poolman
author_facet	Łukasz Syga Dian Spakman Christiaan M. Punter Bert Poolman
author_sort	Łukasz Syga
title	Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
title_short	Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
title_full	Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
title_fullStr	Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
title_full_unstemmed	Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
title_sort	method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/ad83b3b67e6f4a94848e1347944bf434
work_keys_str_mv	AT łukaszsyga methodforimmobilizationoflivingandsyntheticcellsforhighresolutionimagingandsingleparticletracking AT dianspakman methodforimmobilizationoflivingandsyntheticcellsforhighresolutionimagingandsingleparticletracking AT christiaanmpunter methodforimmobilizationoflivingandsyntheticcellsforhighresolutionimagingandsingleparticletracking AT bertpoolman methodforimmobilizationoflivingandsyntheticcellsforhighresolutionimagingandsingleparticletracking
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Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking

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