Chapter

Far-Field Optical Nanoscopy

Volume 14 of the series Springer Series on Fluorescence pp 87-110

Date:

Single-Molecule Photocontrol and Nanoscopy

  • Matthew D. LewAffiliated withDepartment of Chemistry, Stanford UniversityDepartment of Electrical Engineering, Stanford University
  • , Steven F. LeeAffiliated withDepartment of Chemistry, Stanford UniversityDepartment of Chemistry, University of Cambridge
  • , Michael A. ThompsonAffiliated withDepartment of Chemistry, Stanford University
  • , Hsiao-lu D. LeeAffiliated withDepartment of Chemistry, Stanford University
  • , W. E. MoernerAffiliated withDepartment of Chemistry, Stanford University Email author 

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Abstract

Fluorescence microscopy is a ubiquitous tool in biological studies, but fundamental diffraction limits its resolution to ~200 nm for visible light. To overcome this physical limit, but still retain the advantages of far-field noninvasive fluorescence imaging, single-molecule photocontrol has been utilized to achieve optical nanoscopy. Superlocalization, combined with photocontrol of single molecules, allows individual molecules to be localized to precisions of tens of nanometers as part of a larger biological structure, thereby achieving super-resolution. Photoactivation, photoswitching, and photoinduced blinking are all methods of photocontrol, and critical characterization and performance parameters of photocontrollable fluorophores are discussed. We describe two classes of small molecules for use in photoactivation (azido-dicyanomethylenedihydrofuran molecules) and photoswitching (Cy3–Cy5 covalent heterodimer) studies. Furthermore, the use of the first-reported photoswitchable fluorescent protein, enhanced yellow fluorescent protein (eYFP), is also discussed for photoswitching and for photoinduced blinking experiments. Importantly, all of these methods of photocontrol have demonstrated remarkable usefulness in super-resolution studies of structures in living cells.

Keywords

Photoactivation Photoswitching Photoinduced blinking Single-molecule Super-resolution microscopy