Single-Molecule Photocontrol and Nanoscopy

  • Matthew D. Lew
  • Steven F. Lee
  • Michael A. Thompson
  • Hsiao-lu D. Lee
  • W. E. Moerner
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 14)


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.


Photoactivation Photoswitching Photoinduced blinking Single-molecule Super-resolution microscopy 



We warmly thank former Moerner laboratory members Julie Biteen, Nick Conley, and Sam Lord for their contributions to portions of the experiments shown here and the laboratory of Prof. Lucy Shapiro and Jerod Ptacin for C. crescentus cell lines. The work on DCDHF molecules would not have been possible without Prof. Robert J. Twieg’s group at Kent State University who synthesized the molecules. This work was supported in part by the National Institutes of Health Roadmap for Medical Research Grant No. 1P20-HG003638, and by Grant Number R01GM085437 from the National Institute of General Medical Sciences. MDL was supported in part by a National Science Foundation Graduate Research Fellowship and a 3Com Corporation Stanford Graduate Fellowship. MAT was supported in part by a National Science Foundation Graduate Research Fellowship and by a Bert and DeeDee McMurtry Stanford Graduate Fellowship.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Matthew D. Lew
    • 1
    • 2
  • Steven F. Lee
    • 1
    • 3
  • Michael A. Thompson
    • 1
  • Hsiao-lu D. Lee
    • 1
  • W. E. Moerner
    • 1
  1. 1.Department of ChemistryStanford UniversityStanfordUSA
  2. 2.Department of Electrical EngineeringStanford UniversityStanfordUSA
  3. 3.Department of ChemistryUniversity of CambridgeCambridgeUK

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