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2 - One Molecule, Two Molecules, Red Molecules, Blue Molecules

Methods for Quantifying Localization Microscopy Data

from Part I - Super-Resolution Microscopy and Molecular Imaging Techniques to Probe Biology

Published online by Cambridge University Press:  05 May 2022

By
Gaetan G. Herbomel  and
George H. Patterson
Edited by
Krishnarao Appasani  and
Raghu Kiran Appasani
Krishnarao Appasani
Affiliation:
GeneExpression Systems, Inc.
Raghu Kiran Appasani
Affiliation:
Psychiatrist, Neuroscientist, & Mental Health Advocate
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Summary

Improvement of super-resolution microscopy in the last decade has led to the development of methods such as stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM), which modulate the excitation light to break the diffraction limit, or methods such as photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM), which utilize the photoswitching properties of fluorescent molecules to enable precise localization of single molecules (Patterson, 2009). In this chapter, we will focus on PALM/STORM methods, which rely on similar principles and instrumentation. Namely, these acquire a series of images of fields of single molecules followed by subsequent image analysis to localize the molecules to much higher precision than their diffraction limited signals. Importantly, thousands or millions of molecules are identified and used to reconstruct the super-resolution images (Betzig et al., 2006; Hess et al., 2006; Rust et al., 2006; Heilemann et al., 2008; van de Linde et al., 2009; Kamiyama and Huang, 2012).

Type
Chapter
Information
Single-Molecule Science
From Super-Resolution Microscopy to DNA Mapping and Diagnostics
 , pp. 20 - 37
Publisher: Cambridge University Press
Print publication year: 2022

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