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Microscopy Reference Library

Superresolution Structured Illumination Microscopy (SR-SIM)

Lateral resolution can be increased over the classical Abbe limit by a factor of two (approximately 100 to 120 nanometers) in structured illumination without discarding any emission light using laser-generated spatially structured illumination coupled to a widefield fluorescence microscope in what is termed Superresolution (SR) SIM. Linear SR-SIM wavefronts render normally inaccessible high-resolution information available in the form of moiré fringes that contain harmonic frequencies from the specimen that are not available in conventional fluorescence microscopy. Thus, if two fine patterns are superposed in a multiplicative fashion, a beat pattern (moiré fringes) will appear in their product. In this case, one of the patterns is the spatial fluorophore distribution in the specimen and the other is the structured excitation light intensity.

Gustafsson, M. G. L.

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.  Journal of Microscopy 198: 82-87 (2000).  An elegant discussion of structured illumination coupled to demonstrations of the high resolution capabilities exhibited by the technique. The author provides an impressive display of enhanced resolution using stress fibers forming the actin cytoskeleton at the edge of a cultured cell.

Gustafsson, M. G. L.

Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution.  Proceedings of the National Academy of Sciences, USA 102: 13081-13086 (2005).  Experimental demonstration of the high resolution afforded by structured illumination coupled to laser illumination. The concept is thoroughly described and experimentally tested using fluorescent beads.

Fedosseev, R., Belyaev, Y., Frohn, J. and Stemmer, A.

Structured light illumination for extended resolution in fluorescence microscopy.  Optics and Lasers in Engineering 43: 403-414 (2005).  A discussion of how structured illumination can extend optical resolution due to spatial frequencies beyond the classical cut-off frequency being brought into the microscope passband by frequency mixing, termed harmonic excitation light microscopy (HELM).

Gustafsson, M. G. L., Shao, L., Carlton, P. M., Wang, C. J. R., Golubovskaya, I. N., Cande, W. Z., Agard, D. A. and Sedat, J. W.

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.  Biophysical Journal 94: 4957-4970 (2008).  A brilliant description of how structured illumination can be applied in three dimensions to double the axial as well as lateral resolution in order to provide true optical sectioning. The authors give a complete theoretical description with impressive demonstrations using fluorescent beads and stained fixed specimens.

Schermelleh, L., Carlton, P. M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M. C., Agard, D. A., Gustafsson, M. G. L., Leonhardt, H. and Sedat, J. W.

Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.  Science 320: 1332-1336 (2008).  The authors describe the application of three-dimensional structured illumination microscopy (3D-SIM) to the observation of chromatin, nuclear lamina, and the nuclear pore complex using multiple fluorophores in fixed cells.

Beck, M., Aschwanden, M. and Stemmer, A.

Sub-100-nanometre resolution in total internal reflection fluorescence microscopy.  Journal of Microscopy 232: 99-105 (2008).  A unique effort that combines total internal reflection fluorescence microscopy with structured illumination to enable widefield imaging with a lateral resolution beneath 100 nanometers. The authors discuss instrument configuration for what is termed harmonic excitation light microscopy (HELM).

Carlton, P. M.

Three-dimensional structured illumination microscopy and its application to chromosome structure.  Chromosome Research 16: 351-365 (2008).  Professor Carlton reviews the exploration of chromosome structure with high-resolution SIM using three-dimensional approaches. Basics of the technique and practical considerations are described, as well as applications for qualitative evaluation of chromosome structure.

Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. and Gustafsson, M. G. L.

Super-resolution video microscopy of live cells by structured illumination.  Nature Methods 6: 339-342 (2009).  The authors introduce a high-speed structured illumination microscope that is capable of lateral resolution of approximately 100 nanometers at greater than 10 frames per second for several hundred time points. Video imaging of tubulin and kinesin dynamics in living cells was demonstrated.

Lemmer, P., Gunkel, M., Baddeley, D., Kaufmann, R., Urich, A., Weiland, Y., Reymann, J., Mueller, P., Hausmann, M. and Cremer, C.

SPDM: Light microscopy with single-molecule resolution at the nanoscale.  Applied Physics B, Lasers and Optics 93: 1-12 (2008).  Application of spectral precision distance microscopy (SPDM) to spatially modulated illumination using photoconvertable fluorophores. The authors report an effective optical resolution of approximately 20 nanometers in the lateral plane and 50 nanometers in the axial direction.

Hirvonen, L. M., Wicker, K., Mandula, O. and Heintzmann, R.

Structured illumination microscopy of a living cell.  European Biophysics Journal 38: 807-812 (2009).  Dr. Heintzmann and associates utilize a spatial light modulator to generate high-resolution structured excitation illumination patterns for the purposes of examining slowly moving entities in living cells. Details of the instrument, specimen preparation, data reconstruction, and imaging results are discussed.