Abstract
Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.
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References
Galbraith JA, Galbraith CG (2011) Super-resolution microscopy at a glance. J Cell Sci 3:247–255
Taraska JW (2019) A primer on resolving the nanoscale structure of the plasma membrane with light and electron microscopy. J Gen Physiol 151:974–985
Blanchion L, Boujeema-Paterski R, Sykes C, Plastino J (2014) Actin dynamics architecture and mechanics in cell motility. Physiol Rev 94:235–263
Svitkina T (2018) The actin cytoskeleton and actin-based motility. Cold Spring Harb Perspect Biol 10:a018627
Sheppard CJ, Mehta SB, Heintzmann R (2013) Superresolution by image scanning microscopy using pixel reassignment. Opt Lett 38:2889–2892
Tinevez JY, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, Bednarek SY, Shorte SL, Eliceiri KW (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90
Mary H, Brouhard GJ (2019) Kappa (K): analysis of curvature in biological image data using B-splines. bioRxiv https://doi.org/10.1101/852772
Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB (2000) Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49:424–430
Trexler AJ, Taraska JW (2017) Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells. Cell Calcium 67:1–10
Orci L, Gabbay KH, Malaisse WJ (1972) Pancreatic beta cell web: its possible role in insulin secretion. Science 175:1128–1130
Orci L, Thorens B, Ravazzola M, Lodish HF (1989) Localization of pancreatic beta cell glucose transporter to specific plasma membrane domains. Science 245:295–297
Bénichou O, Moreau M, Suet PH, Voituriez R (2007) Intermittent search process and teleportation. J Chem Phys 126:234109
Steyer JA, Almers W (2001) A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2:268–275
Axelrod D (2013) Evanescent excitation and emission in fluorescence microscopy. Biophys J 104:1401–1409
Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87
Kner P, Chhun BB, Griffis ER, Winoto L, Gustafsson MG (2009) Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6:339–342
Korobchevskaya K, Lagerholm CB, Colin-York H, Fritzsche M (2017) Exploring the potential of Airyscan microscopy for live cell imaging. Photo-Dermatology 4:41
Sarder P, Nohorai A (2006) Deconvolution methods for 3-D fluorescence microscopy images. IEEE Signal Process Mag 23:32–45
Huff J (2015) The Airyscan detector from Zeiss: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat Methods:12, i-ii
Sheppard CJ (1988) Superresolution in confocal imaging. Optik 80:53–54
Bertero M, Brianzi P, Pike ER (1999) Super-resolution in confocal scanning microscopy. Inverse Probl 3:195–212
Li D, Shao L, Chen BC, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA III, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E (2015) Advanced imaging extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349:aab3500
Sato Y, Nakajima S, Shiraga N, Atsumi H, Yoshida S, Koller T, Gerig G, Kikinis R (1998) Three-dimensional multi-scale filter for segmentation and visualization of curvilinear structures in medical images. Med Image Anal 2:143–168
Young LJ, Ströhl F, Kaminski CF (2016) A guide to structured illumination TIRF microscopy at high speed with multiple colors. J Vis Exp 111:e53988
Acknowledgments
The authors would like to thank Dr. Teng-Leong Chew, Director of the Advanced Imaging Center, and his team at Howard Hughes Medical Institute (https://www.aicjanelia.org/) for technical support with the TIRF-SIM imaging system. We would also like to thank Dr. Tanay Desay from Carl Zeiss Microscopy, LLC, for technical support with the LSM980 with Airyscan 2 and Drs. George G. Holz and Oleg Chepurny from SUNY Upstate Medical University for advice with the INS-1 832/13 cell culture .
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Wöllert, T., Langford, G.M. (2022). Super-Resolution Imaging of the Actin Cytoskeleton in Living Cells Using TIRF-SIM. In: Gavin, R.H. (eds) Cytoskeleton . Methods in Molecular Biology, vol 2364. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1661-1_1
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DOI: https://doi.org/10.1007/978-1-0716-1661-1_1
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