Washington University in St Louis

The Preston M. Green Department of
Electrical & Systems Engineering

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Super-resolution fluorescence microscopy


Many of the important activities and processes in biology, technology, and materials science occur at microscopic and nanoscopic length scales. However, wave physics limits light microscopes from resolving detail much smaller than the wavelength of light (~500 nm in the visible). Super-resolution imaging allows light microscopes to overcome this fundamental limitation, enabling these minimally-invasive instruments to see small details within living bacteria and animal cells.

Adapted from Guilaume Paumier and licensed under the CC BY-SA 2.5 license. Biological and technological size scales. Adapted from http://commons.wikimedia.org/wiki/File:Biological_and_technological_scales_compared-en.svg and licensed under CC BY-SA 2.5.

Dr. Eric Betzig, Prof. Stefan W. Hell, and Prof. W. E. Moerner were jointly awarded the Nobel Prize in Chemistry 2014 for the development of super-resolved fluorescence microscopy. Their lectures are a good introduction to the history and concepts behind this technology.


Conventional microscope image that is unable to resolve microtubule structures within a cell
A normal microscope is unable to resolve all of the microtubules within this mammalian cell.

Single fluorescent molecules can be used as molecular beacons that report the location of biomolecules within cells, much like using Christmas lights to light up specific leaves of a Christmas tree. The structures within cells are so small that normal microscopes blur all of the molecules together, preventing us from resolving them from one another.

Alexa Fluor 647 molecules blinking within a mammalian cell.

However, if we can control the emission of those fluorescent molecules, by making them blink in time for example, then we can resolve those molecules from one another. There are many photochemical means of controlling fluorescent molecules, and many types of molecules can blink very quickly (~100 Hz or 100 times per second).

Super-resolution microscope image resolves the microtubule structures within a cell
A super-resolution microscope can capture the complex structure of microtubules within a mammalian cell.

Once we can separate each molecule from its neighbors by having them blink, then we can measure the position of each molecule using image-processing software. A final reconstructed image of the sample is created by combining all of the position measurements of individual molecules together.

Single-molecule super-resolution microscopy, also termed single-molecule localization microscopy but not to be confused with stimulated emission depletion microscopy (STED), uses blinking single fluorescent molecules and image-processing algorithms in order to create super-resolved images.

Related papers

  1. M. A. Thompson, M. D. Lew, and W. E. Moerner, “Extending microscopic resolution with single-molecule imaging and active control,” Annu. Rev. Biophys. 41, 321 (2012). [Journal]
  2. S. J. Sahl and W. E. Moerner, “Super-resolution fluorescence imaging with single molecules,” Curr. Opin. Struct. Biol. 23, 778-87 (2013). [Journal]
  3. E. Betzig, “Single Molecules, Cells, and Super-Resolution Optics (Nobel Lecture),” Angew. Chemie Int. Ed. 54, 8034-8053 (2015). [Journal]
  4. S. W. Hell, “Nanoscopy with Focused Light (Nobel Lecture),” Angew. Chemie Int. Ed. 54, 8054-8066 (2015). [Journal]
  5. W. E. Moerner, “Single-Molecule Spectroscopy, Imaging, and Photocontrol: Foundations for Super-Resolution Microscopy (Nobel Lecture),” Angew. Chemie Int. Ed. 54, 8067-8093 (2015). [Journal]

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Optics and microscopy

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