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Nanoscale cellular imaging with scanning angle interference microscopy

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Christopher DuFort*,{, Matthew Paszek{,} *Department of Surgery, University of California, San Francisco, California, Department of Orthopaedic Surgery, University of California, San Francisco, California, { School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, } Kavli Institute at Cornell for Nanoscale Science, Ithaca, New York, {

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CHAPTER OUTLINE Introduction ............................................................................................................ 236 Superresolution Optical Imaging ............................................................................. 236 Theory of SAIM ...................................................................................................... 238 13.1 Experimental Methods and Instrumentation......................................................241 13.1.1 Microscope and Instrumentation ................................................ 241 13.1.2 Preparation of Reflective Substrates........................................... 242 13.1.3 Selection of Fluorescent Probes ................................................. 242 13.1.4 Cell Culture and Transfection .................................................... 243 13.1.5 Immunolabeling of Samples ...................................................... 244 13.1.6 Microscope Calibration and Configuration ................................... 246 13.1.7 Image Acquisition .................................................................... 247 13.2 Image Analysis and Reconstruction.................................................................250 Conclusion ............................................................................................................. 250 Acknowledgments ................................................................................................... 251 References ............................................................................................................. 251

Abstract Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an Methods in Cell Biology, Volume 123, ISSN 0091-679X, http://dx.doi.org/10.1016/B978-0-12-420138-5.00013-6 © 2014 Elsevier Inc. All rights reserved.

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emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5–10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (

Nanoscale cellular imaging with scanning angle interference microscopy.

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved...
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