Information
A brief introduction to each of the main techniques is given below:
3-D Structured Illumination Microscopy (3D-SIM)
3D-SIM illuminates the sample with a periodic pattern that interacts with small features in the sample to generate Moiré images with higher spatial features. An algorithm is used to reconstruct an image with an increase in resolution in both xy and z. SIM can be used with most commonly used fluorophores. It is suitable for multi colour imaging and 3D sectioning can be used to obtain images of whole cells. The resolution is improved ~two-fold in all dimensions over conventional optical microscopy methods giving a lateral resolution of ~100 nm, and a z resolution of ~ 250 nm.
Stimulated Emission Depletion Microscopy (STED)
STED microscopy uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit. It employs two different lasers, a conventional excitation laser and a toroidal or doughnut-shaped stimulated emission depletion (STED) laser. The STED laser deactivates the fluorescence outside a small central zone, reducing the effective excitation area. Thus the fluorescence signal is observed only from a small region. The resolution in xy that can be achieved is ~80 nm for commercial systems, with ~20 nm achieved experimentally. There is no improvement in z-resolution.
Photo-Activated Localisation Microscopy (PALM) and Stochastic Optical Reconstruction (STORM) Microscopy
PALM and STORM rely on photoactivation, photoconversion, photobleaching or blinking processes to turn fluorescent molecules on and off. Because photoactivation is a stochastic process, only a few, well separated molecules "turn on." Each ‘emitter’ is individually captured in an image without overlap. Two-dimensional Gaussian functions are fit to determine the centre of the peak for each single emitter. These molecules are then turned off and a new set turned on until the entire image is captured. The “resolution" for this technique (where resolution is defined as the distance between objects that can be reliably measured rather than the true optical resolution) reaches ~10 nm. It is generally implemented in the Total Internal Reflection mode, which means it can be used to look at events close to the microscope cover slip.
Coherent Anti-Stokes Raman Scattering (CARS) Microscopy
CARS microscopy provides high spatial resolution along with chemical specificity. CARS is sensitive to the vibrational signatures of molecules that are used in Raman spectroscopy, typically the nuclear vibrations of chemical bonds. CARS employs multiple photons to address the molecular vibrations, and produces a signal in which the emitted waves are coherent with one another.
Scanning Near Field Optical Microscopy (SNOM)
Perhaps the oldest “super-resolution” technique, SNOM provides high spatial resolution by limiting the area of photoexcitation (or signal collection) through the use of an optical fibre in a scanning probe configuration analogous to atomic force microscopy. Optical fibres can be drawn down to reduce the effective aperture through which the light exits or enters the fibre to a few tens of nanometres. The fibre tip is scanned over a sample (or the sample is scanned relative to the fibre tip) while a constant, close distance between the sample and tip is maintained. Imaging is then in the near-field. If this distance is very small, the divergence of the light emerging from the tip is small and spatial resolution is retained.
Total Internal Reflection Fluorescence (TIRF) Microscopy
TIRF microscopy provides high spatial resolution in the z-plane using the exponentially decaying evanescent field induced by total internal reflection of light at the interface of two media of differing refractive indices. This “wide-field” method, while not providing resolution enhancement in the x-y plane (lateral), provides resolution on the tens of nanometres in the z- (axial) plane and can provide sharper images than conventional wide-field methods by not inducing fluorescence signal outside the penetration of the evanescent field.
Recent reviews
- Esposito, A., S. Schlachter, G.S. Schierle, A.D. Elder, A. Diaspro, F.S. Wouters, C.F. Kaminski, A.I. Iliev, 2009. Quantitative fluorescence microscopy techniques. Methods Mol Biol 586, 117-42
- Huang, B., 2010. Super-resolution optical microscopy: multiple choices. Curr Opin Chem Biol 14, 10-4.
- Langhorst, M.F., J. Schaffer, B. Goetze, 2009. Structure brings clarity: structured illumination microscopy in cell biology. Biotechnol J 4, 858-65.
- Patterson, G., M. Davidson, S. Manley, J. Lippincott-Schwartz, 2010. Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61, 345-67.
- Swedlow, J.R., 2010. Advanced hardware and software tools for fast multidimensional imaging of living cells. Proc Natl Acad Sci U S A. www.pnas.org/cgi/doi/10.1073/pnas.1010043107
- Schermelleh, L., R. Heintzmann, H. Leonhardt, 2010. A guide to super-resolution fluorescence microscopy. J Cell Biol 190, 165-75.
- van Zanten, T.S., A. Cambi, M.F. Garcia-Parajo, 2010. A nanometer scale optical view on the compartmentalization of cell membranes. Biochim Biophys Acta 1798, 777-87.
- Wessels, J.T., K. Yamauchi, R.M. Hoffman, F.S. Wouters, 2010. Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo. Cytometry A 77, 667-76.
- Zhuang, X., 2009. Nano-imaging with Storm. Nat Photonics 3, 365-367.
- Report from the Super Resolution Microscopy workshop held at the Melbourne Convention Centre on 1st October 2010.
Key References
PALM/ STORM
- Bates, M., B. Huang, G.T. Dempsey, X. Zhuang, 2007. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749-53.
- Betzig, E., G.H. Patterson, R. Sougrat, O.W. Lindwasser, S. Olenych, J.S. Bonifacino, M.W. Davidson, J. Lippincott-Schwartz, H.F. Hess, 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642-5.
- Gould, T.J., S.T. Hess, 2008. Chapter 12: Nanoscale biological fluorescence imaging: breaking the diffraction barrier. Methods Cell Biol 89, 329-58.
- Gould, T.J., M.S. Gunewardene, M.V. Gudheti, V.V. Verkhusha, S.R. Yin, J.A. Gosse, S.T. Hess, 2008. Nanoscale imaging of molecular positions and anisotropies. Nat Methods 5, 1027-30
- Gould, T.J., V.V. Verkhusha, S.T. Hess, 2009. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat Protoc 4, 291-308.
- Hess, S.T., T.P. Girirajan, M.D. Mason, 2006. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91, 4258-72.
- Hess, S.T., T.J. Gould, M.V. Gudheti, S.A.
- Hess, S.T., T.J. Gould, M. Gunewardene, J. Bewersdorf, M.D. Mason, 2009. Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy. Methods Mol Biol 544, 483-522.
- Hess, S.T., 2009. Red lights, camera, photoactivation! Nat Methods 6, 124-5.
- Huang, B., W. Wang, M. Bates, X. Zhuang, 2008. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810-3.
- Huang, B., S.A. Jones, B. Brandenburg, X. Zhuang, 2008. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5, 1047-52.
- Huang, B., M. Bates, X. Zhuang, 2009. Super-resolution fluorescence microscopy. Annu Rev Biochem 78, 993-1016.
- Ji, N., H. Shroff, H. Zhong,
- Juette, M.F., T.J. Gould, M.D. Lessard, M.J. Mlodzianoski, B.S. Nagpure, B.T. Bennett, S.T. Hess, J. Bewersdorf, 2008. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5, 527-9.
- Owen, D.M., C. Rentero, J. Rossy, A. Magenau, D. Williamson, M. Rodriguez, K. Gaus, 2010. PALM imaging and cluster analysis of protein heterogeneity at the cell surface. J Biophotonics 3, 446-54.
- Rust, M.J., M. Bates, X. Zhuang, 2006. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793-5.
- Subach, F.V., G.H. Patterson, M. Renz, J. Lippincott-Schwartz, V.V. Verkhusha, 2010. Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J Am Chem Soc 132, 6481-91.
- Tang, J., J. Akerboom, A. Vaziri, L.L. Looger, C.V. Shank, 2010. Near-isotropic 3D optical nanoscopy with photon-limited chromophores. Proc Natl Acad Sci U S A 107, 10068-73.
3D-SIM
- Carlton, P. M., (2008) Three-dimensional structured illumination microscopy and its application to chromosome structure. Chromosome Res 16: 351-365.
- Gustafsson, M. G., (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198: 82-87.
- Gustafsson, M. G., L. Shao, P. M. Carlton, C. J. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard & J. W. Sedat, (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94: 4957-4970.
- Hanssen, E., P. Carlton, S. Deed, N. Klonis, J. Sedat, J. DeRisi & L. Tilley, (2010) Whole cell imaging reveals novel modular features of the exomembrane system of the malaria parasite, Plasmodium falciparum. Int J Parasitol 40: 123-134.
- Heintzmann, R., and C. Cremer. 1999. Lateral modulated excitation microscopy: Improvement of resolution by using a diffraction grating. Proc. SPIE. 3568:185-195.
- Heintzmann, R., T.M. Jovin, and C. Cremer. 2002. Saturated patterned excitation microscopy—a concept for optical resolution improvement. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19:1599-1609.
- Heintzmann, R. & G. Ficz, (2007) Breaking the resolution limit in light microscopy. Methods Cell Biol 81: 561-580.
- Hirvonen, L. M., K. Wicker, O. Mandula & R. Heintzmann, (2009) Structured illumination microscopy of a living cell. Eur Biophys J 38: 807-812.
- Kner, P., B. B. Chhun, E. R. Griffis, L. Winoto & M. G. Gustafsson, (2009) Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6: 339-342.
- Schermelleh, L., P. M. Carlton, S. Haase, L. Shao, L. Winoto, P. Kner, B. Burke, M. C. Cardoso, D. A. Agard, M. G. Gustafsson, H. Leonhardt & J. W. Sedat, (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320: 1332-1336.
STED
- Egner, A. & S. W. Hell, (2005) Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol 15: 207-215.
- Han, K.Y., K.I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, S.W. Hell, 2009. Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light. Nano Lett 9, 3323-9.
- Kasper, R., B. Harke, C. Forthmann, P. Tinnefeld, S.W. Hell, M. Sauer, 2010. Single-Molecule STED Microscopy with Photostable Organic Fluorophores. Small 6, 1379-1384
- Lauterbach, M.A., J. Keller, A. Schonle, D. Kamin, V. Westphal, S.O. Rizzoli, S.W. Hell, 2010. Comparing video-rate STED nanoscopy and confocal microscopy of living neurons. J Biophotonics (in press).
- Van de Linde, S., M. Sauer, M. Heilemann, 2008. Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores. J Struct Biol 164, 250-4.
Other methods
- Dertinger, T., R. Colyer, G. Iyer,
- Le Gros, M.A., G. McDermott, M. Uchida, C.G.
- Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH. 2008. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322: 1065-1069.
Super-Resolution Optical Microscopy Sites
Nature Microscopy Special
http://www.nature.com/news/specials/microscopy/index.htmlhttp://www.nature.com/nmeth/focus/moy2008/index.html
http://www.nature.com/nmeth/journal/v6/n1/abs/nmeth.f.234.html
Super resolution microscopy: articles & links
http://www.superresolutionmicroscopy.com/http://zeiss-campus.magnet.fsu.edu/referencelibrary/superresolution.html
http://www.andor.com/learning/applications/Super-Res_Microscopy/
http://www.zeiss.de/C12567BE0045ACF1/Contents-Frame/7EFC4393ABB349F9C125758D00210C72