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Microscopy Reference Library

Light Sheet Microscopy

Light sheet microscopy, often referred to as single plane illumination microscopy (SPIM), is a rapidly emerging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with impressive resolution. Unlike the conventional techniques of widefield and confocal fluorescence microscopy, the light sheet technique illuminates on the region surrounding the focal plane of the detection objective in a twin objective configuration (where the objectives are juxtaposed at 45-degrees). When compared to conventional microscopy, light sheet methods exhibit reduced photobleaching and lower phototoxicity, and often enable far more scans per specimen. By rotating the specimen, the technique can image virtually any plane with multiple views obtained from different angles. Thus, light sheet microscopy is ideal for examining of both large (animals) and small (cells) specimens labeled with fluorescent proteins and other fluorophores.

Santi, P. A.

Light sheet fluorescence microscopy: A reviewJournal of Histochemistry and Cytochemistry 59: 129-138 (2011).  Dr. Santi introduces a nicely composed review article that covers the basic principles of light sheet microscopy that includes a historical perspective, instrumentation requirements, and details about how to process specimens for imaging.

Keller, P. J., Schmidt, A. D., Santella, A., Khairy, K., Bao, Z., Wittbrodt, J. and Stelzer, E. H. K.

Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopyNature Methods 7: 637-642 (2010).  The authors describe an advanced scanning light sheet technology that employs structured illumination patterns with continuously adjustable frequencies. The resulting images discriminate between scattered background noise from the signal fluorescence.

Huisken, J. and Stainier, D. Y. R.

Selective plane illumination microscopy techniques in developmental biologyDevelopment 136: 1963-1975 (2009).  An excellent review of selective plane illumination microscopy that discusses optical sectioning, introduces a glossary of terms, and describes in detail the concept and necessary components. Also described are sample preparation and advanced techniques.

Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. and Stelzer, E. H. K.

Optical sectioning deep inside live embryos by selective plane illumination microscopyScience 305: 1007-1009 (2004).  One of the foremost early review articles on light sheet microscopy demonstrating the imaging of live specimens. Termed Selective Plane Illumination Microscopy (SPIM) by the authors, the article describes instrumentation and techniques used to image living Medaka (Japanese killifish) embryos.

Planchon, T. A., Gao, L., Milkie, D. E., Davidson, M. W., Galbraith, J. A., Galbraith, C. G. and Betzig, E.

Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illuminationNature Methods 8: 417-423 (2011).  Eric Betzig and collaborators introduce light sheet microscopy using scanned Bessel beams in conjunction with structured illumination and two-photon excitation to create thinner sheets (less than one-half micrometer). The technique is better suited for three-dimensional imaging of single cells.

Greger, K., Swoger, J. and Stelzer, E. H. K.

Basic building units and properties of a fluorescence single plane illumination microscopeReview of Scientific Instruments 78: 023705-7 (2007).  A thorough discussion of the important concepts underlying construction of a light sheet microscope. Included are descriptions on the efficient use of fluorophores, phototoxicity, optical sectioning, and axial resolution.

Dodt, H. U., Leischner, U., Schierloh, a., Jaehrling, N., Mauch, C. P., Deininger, K., Deussing, J. M., Edner, M., Zieglgaensberger, W. and Becker, K.

Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brainNature Methods 4: 331-336 (2007).  Introducing the concept of light sheet microscopy as ultramicroscopy in clear tissue, the authors demonstrate a powerful technique for optical sectioning of fixed brain tissue expressing fluorescent proteins.

Keller, P. J. and Stelzer, E. H. K.

Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopyCurrent Opinion in Neurobiology 18: 624-632 (2008).  An excellent introductory review article on light sheet microscopy by two of experts in the field. Discussed are basic instrumental principles, illumination efficiency, optical sectioning, resolution, signal-to-noise, and imaging speed.

Keller, P. J., Schmidt, A. D., Wittbrodt, J. and Stelzer, E. H. K.

Reconstruction of zebrafish early embryonic development by scanned light sheet microscopyScience 322: 1065-1069 (2008).  A remarkable investigation of nucleus localization and movement in zebrafish embryos over their first 24 hours of development. The authors were able to image specimens at 1.5 billion voxels per minute to generate a comprehensive database.

Reynaud, E. G., Krzic, U., Greger, K. and Stelzer, E. H. K.

Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamageHFSP Journal 2: 266-275 (2008).  A comparison of digital light sheet microscopy with conventional widefield and confocal fluorescence techniques. The authors describe optical sectioning, photobleaching, and phototoxicity differences between the techniques.