Modern light microscopes make use of fluorescent probes to label specific structures inside cells, like DNA. NPL now uses super-resolution microscopes to bypass the diffraction limit of light and see near molecular-level detail inside cells, with up to 10 times more resolving power than a standard optical microscope.
Light microscopes have a resolution limit of around 250 nanometres (nm), which means anything smaller will appear blurred. Any improvements in microscope resolution will give new insights into molecular details of cells and improve our understanding and treatment of diseases. Recently a number of different approaches, referred to as super-resolution, have been developed that can bypass the resolution limit by a factor of 2 or more. At NPL, we have developed our own versions of two of these techniques which offer a complementary capability: localisation microscopy (STORM) and structured illumination microscopy (SIM).
STORM offers the highest resolution with the potential for measuring the number of molecules in a sample. Some of the limitations of this technique are that its image acquisition time is relatively slow, which limits its practical uses to fixed samples, and only a restricted range of fluorescent dyes and proteins are suitable for sample labelling.
SIM is compatible with many more fluorescent dyes and proteins. It is also a far quicker technique with image acquisition times only limited by the brightness of the sample. This makes it applicable in both fixed and live samples. Compared with localisation microscopy the resolution enhancement is less impressive, however, it is still a two-fold improvement over diffraction limited fluorescence microscopy techniques.
|Technique||Lateral resolution (nm)||Typical image acquisition time|
|Standard fluorescence microscopy||250||0.5-10 seconds|
- Correlative super resolution using dSTORM and SIM
- STORM-ing through the diffraction limit
- Application of Super-Resolution Imaging to the Endocytic Pathway
Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders
Westmoreland, D., Shaw, M., Grimes, W., Metcalf, D. J., Burden, J. J., Gomez, K., Knight, A.E. and Cutler, D.F.
J. Thromb. Haemost., 13:1-11 (2016)
A two-tier Golgi-based control of organelle size underpins the functional plasticity of endothelial cells
Ferraro F, Kriston-Vizi J, Metcalf D J, Martin-Martin B, Freeman J, Burden J J, Westmoreland D, Dyer C E, Knight A E, Ketteler R and Cutler D F
Dev Cell, 29, 292-304 (2014)
TestSTORM: simulator for optimizing sample labeling and image acquisition in localization based super-resolution microscopy
Sinkó J, Kákonyi R, Rees E, Metcalf D, Knight A E, Kaminski C F, Szabó G and Erdélyi M
Biomed Opt Express, 5, 778-787 (2014)
Elements of image processing in localization microscopy
Eric J Rees, Miklos Erdelyi, Gabriele S Kaminski Schierle, Alex Knight and Clemens F Kaminski
Journal of Optics, 15 (9):094012 (2013)
Test Samples for Optimizing STORM Super-Resolution Microscopy
Metcalf, D. ., Edwards, R., Kumarswami, N., Knight, A.
J. Vis. Exp., 79, e50579, doi:10.3791/50579 (2013)
Correcting chromatic offset in multicolor super-resolution localization microscopy
Erdelyi M, Rees E, Metcalf D, Schierle GS, Dudas L, Sinko J, Knight AE, Kaminski CF
Opt Express, 21, 10978-10988 (2013)
Blind assessment of localisation microscope image resolution
Rees E, Erdelyi M, Pinotsi D, Knight AE, Metcalf D, Kaminski CF
Optical Nanoscopy, 1.12, 2012
Polarization effects on contrast in structured illumination microscopy
O'Holleran K, Shaw MJ
Opt. Lett., 2012, 37, 4603-4605
Single Molecule Pointillism
Erdelyi M, Metcalf D
Imaging & Microscopy (2012)
In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging
Schierle GSK, van de Linde S, Erdelyi M, Esbjorner EK, Klein T, Rees T, Bertoncini CW, Dobson CM, Sauer M, Kaminski CF
J. Am. Chem. Soc., 133 (33), pp 12902-12905 (2011)
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