Abstract
Platinum replicas of unroofed mammalian cells can be imaged with a transmission electron microscope (TEM) to produce high contrast, high resolution images of the structure of the cytoplasmic side of a plasma membrane. A complementary approach, super-resolution fluorescence localization microscopy, can be used to localize labeled molecules with better than 20 nm precision in cells. Here, we describe a correlative method that couples these two techniques and produces images where localization microscopy data can be used to highlight specific proteins of interest within the structural context of the platinum replica TEM image. This combined method is uniquely suited to investigate the nanometer-scale structural organization of the plasma membrane and its associated organelles and proteins.
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References
Moor H, Mühlethaler K (1963) Fine structure in frozen-etched yeast cells. J Cell Biol 17(3):609–628
Lung B (1968) Whole-mount electron microscopy of chromatin and membranes in bull and human sperm heads. J Ultrastruct Res 22(5):485–493
Heuser J, Keen J (1988) Deep-etch visualization of proteins involved in clathrin assembly. J Cell Biol 107(3):877–886
Svitkina T (2015) Imaging cytoskeleton components by electron microscopy. Meth Mol Biol (Clifton, NJ) 1365:99–118
Heuser J (2000) The production of ‘cell cortices’ for light and electron microscopy. Traffic 1(7):545–552
Sochacki KA, Shtengel G, van Engelenburg SB, Hess HF, Taraska JW (2014) Correlative super-resolution fluorescence and metal-replica transmission electron microscopy. Nat Methods 11(3):305–308
Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci 106(9):3125–3130
Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1):36–51
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71–76
Shtengel G, Wang Y, Zhang Z, Goh WI, Hess HF, Kanchanawong P (2013) Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM. Methods Cell Biol 123:273–294
Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8(12):1027–1036
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Sochacki, K.A., Taraska, J.W. (2017). Correlative Fluorescence Super-Resolution Localization Microscopy and Platinum Replica EM on Unroofed Cells. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_18
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DOI: https://doi.org/10.1007/978-1-4939-7265-4_18
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7264-7
Online ISBN: 978-1-4939-7265-4
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