Effect of Different Fluorescence Iabelling Methods of the Actin Cytoskeleton for Storm Application
Journal: Journal of Higher Education Research DOI: 10.32629/jher.v5i4.2658
Abstract
The Abbe limit was a major challenge in optical microscopy, making it difficult to observe microscopic biological structures. However, the development of STORM, a new fluorescence localization microscopy technique, has enabled much higher resolution imaging by causing dye molecules to blink under laser light. Advances in computational analysis have further improved image processing and resolution, leading to clearer observations and a better understanding of biological structures. Actin, a common protein in eukaryotic cells, forms F-actin, a filament involved in essential processes like cell division, migration, and muscle contraction. Fluorescence labeling is crucial for visualizing F-actin and other cellular structures, allowing scientists to study these important processes. This project explored two methods to visualize actin structures in Hela cells: phalloidin labeling with Alexa-647 dye and DNA-PAINT using GFP. ThunderSTORM analysis produced normalized images for both methods, and resolution values were calculated using FRC. The results indicated that phalloidin labeling provided higher resolution but lower-quality images, while DNA-PAINT with GFP offered better-quality images but with lower resolution.
Keywords
cell, optical microscopy, flourscence labelling, DNA-PAINT
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[5]Schnitzbauer, J., Strauss, M., Schlichthaerle, T. et al. Super-resolution microscopy with DNA-PAINT. Nat Protoc 12, 1198–1228 (2017). https://news.mit.edu/2016/programmable-rna-vaccines-0704
[6]Pardi, N., Hogan, M., Porter, F. et al. mRNA vaccines — a new era in vaccinology. Nat Rev Drug Discov 17, 261–279 (2018). https://www.abbelight.com/dna-paint/.
[7]Sharonov, A. Hochstrasser, R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006)
[8]Stehr, F., Stein, J., Bauer, J. et al. Tracking single particles for hours via continuous DNA-mediated fluorophore exchange. Nat Commun 12, 4432 (2021). https://oni.bio/storm-microscopy/#:~:text=STORM.
[9]Cox,S.(2015).Super-Resolution Imaging in Living Cells.Developmental Biology,175-81.
[10]Li, Y., Mund, M., Hoess, P. et al. Real-time 3D single-molecule localization using experimental point spread functions. Nat Methods 15, 367–369 (2018). https://doi.org/10.1038/nmeth.4661
[11]Costello,I.(2018).Nanoscale Mapping of Sarcomeric Structure Mechanical Properties. King’s College London.
[12]Koho, S., Tortarolo, G., Castello, M. et al. Fourier ring correlation simplifies image restoration in fluorescence microscopy. Nat Commun 10, 3103 (2019). https://doi.org/10.1038/s41467-019-11024-z
[13]Van Heel, M., Keegstra, W., Schutter, W. Van Bruggen, E. J. F. Arthropod hemocyanin structures studied by image analysis. Life Chem. Rep. Suppl. 1, 69–73 (1982).
[14]Saxton, W. O. Baumeister, W. The correlation averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127–138 (1982).
[15]Wehland, J., Osborn, M., and Weber, K. (1977). Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth[J]. Proc. Natl. Acad. Sci. 74, 5613–5617.
[16]Cooper, J. A. (1987). Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105, 1473–1478.
[17]Melak, M., Plessner, M., and Grosse, R. (2017). Actin visualization at a glance. J. Cell Sci. 130, 525–530. doi: 10.1242/jcs.189068
[18]Aizawa, H., Sameshima, M., and Yahara, I. A. (1997). green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium. Cell Struct. Funct. 22, 335–345. doi: 10.1247/csf.22.335
[19]Nagasaki, A., Kijima, T., Yumoto, T., Imaizumi, M., Yamagishi, A., Kim, H., et al. (2017). The Position of the GFP Tag on Actin Affects the Filament Formation in Mammalian Cells. Cell Struct. Funct. 42, 131–140. doi: 10.1247/csf.17016
Riedl J, Flynn KC, Raducanu A, Gärtner F, Beck G, Bösl M, Bradke F, Massberg S, AszodiA, SixtM, Wedlich-Söldner R. Lifeact mice for studying F-actin dynamics. Nat Methods. 2010 Mar;7(3):168-9. doi: 10.1038/nmeth0310-168. PMID: 20195247
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