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      Cell membrane sample preparation method of combined AFM and dSTORM analysis

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          Abstract

          A major role of cell membranes is to provide an ideal environment for the constituent proteins to perform their biological functions. A deep understanding of the membrane proteins assembly process under physiological conditions is quite important to elucidate both the structure and the function of the cell membranes. Along these lines, in this work, a complete workflow of the cell membrane sample preparation and the correlated AFM and dSTORM imaging analysis methods are presented. A specially designed, angle-controlled sample preparation device was used to prepare the cell membrane samples. The correlated distributions of the specific membrane proteins with the topography of the cytoplasmic side of the cell membranes can be obtained by performing correlative AFM and dSTORM measurements. These methods are ideal for systematically studying the structure of the cell membranes. The proposed method of the sample characterization was not only limited to the measurement of the cell membrane but also can be applied for both biological tissue section analysis and detection.

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          Most cited references24

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          Atomic Force Microscope

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            Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

            Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.
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              High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy.

              Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.
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                Author and article information

                Contributors
                Journal
                Biophys Rep
                Biophys Rep
                BR
                Biophysics Reports
                Biophysics Reports Editorial Office (Beijing China )
                2364-3439
                2364-3420
                31 August 2022
                : 8
                : 4
                : 183-192
                Affiliations
                [1 ] University of Science and Technology of China, Hefei 230027, China
                [2 ] State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
                [3 ] Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, Shandong, China
                Author notes

                Huili Wang contributed equally to this work.

                Article
                br-8-4-183
                10.52601/bpr.2022.220004
                10185485
                49e63fb3-768f-4f75-98fa-5e57e652085a
                © The Author(s) 2022

                This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 31 January 2022
                : 6 April 2022
                Categories
                Method

                atomic force microscopy (afm),super-resolution microscopy (srm),direct stochastic optical reconstruction microscopy (dstorm),cell membrane,combined technique

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