Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.

Autor: Koester AM; Program in Quantitative and Systems Biology, Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon, USA., Szczepaniak M; Program in Quantitative and Systems Biology, Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon, USA., Nan X; Program in Quantitative and Systems Biology, Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon, USA.; Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.
Jazyk: angličtina
Zdroj: Current protocols [Curr Protoc] 2022 Nov; Vol. 2 (11), pp. e618.
DOI: 10.1002/cpz1.618
Abstrakt: Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of probes for DNA-PAINT-ERS Basic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cells Alternate Protocol: Immunostaining of extracted U2OS cells Basic Protocol 3: Super resolution image acquisition and analysis.
(© 2022 Wiley Periodicals LLC.)
Databáze: MEDLINE
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