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Microscopy Reference Library

Structured Illumination for Optical Sectioning

Structured illumination, often referred to as a "poor man's confocal microscope" is emerging as a powerful technique for optical sectioning in widefield microscopy at high resolution. Although current implementations are limited in speed and multi-channel acquisition by the requirement of capturing multiple images, new technological innovations are occurring rapidly in this field. The references listed in this section point to original research reports and review articles that should provide the starting point for a thorough understanding of structured illumination.

Juskaitis, R., Wilson, T., Neil, M. A. A. and Kozubek, M.

Efficient real-time confocal microscopy with white light sources.  Nature 383: 804-806 (1996).  One of the original research reports describing the structured illumination approach. The authors demonstrate a white-light, multiple-point source instrument that can produce images in real time with light efficiencies as high as 50 percent.

Neil, M. A. A., Juskaitis, R. and Wilson, T.

Method of obtaining optical sectioning by using structured light in a conventional microscope.  Optics Letters 22: 1905-1907 (1997).  The original research report on structured illumination that describes projecting a grid pattern onto the specimen at the focal plane. Included is a thorough mathematical description of the technique, microscope configuration, axial response, and images of a pollen grain.

Neil, M. A. A., Juskaitis, R. and Wilson, T.

Real time 3D fluorescence microscopy by two beam interference illumination.  Optics Communications 153: 1-4 (1998).  The demonstration of optical sectioning using structured illumination coupled to a standard widefield microscope. The investigators utilized two interfering beams on the specimen to create a single spatial frequency fringe pattern. The resulting images taken at three spatial positions of the fringe patterns were processed to produce optical sections.

Langhorst, M. F., Schaffer, J. and Goetze, B.

Structure brings clarity: Structured illumination microscopy in cell biology.  Biotechnology Journal 4: 858-865 (2009).  An excellent review on all phases of structured illumination microscopy. The authors discuss basic principles and theoretical concepts, dynamic range, specimen choice, structured illumination for dynamic events, and briefly describe high resolution applications.

Schaefer, L. H., Schuster, D. and Schaffer, J.

Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach.  Journal of Microscopy 216: 165-174 (2004).  The authors describe practical applications of structured illumination microscopy and review potential artifacts. Among the most serious are residual stripe patterns originating from the illumination grating. Suggestions for correction of this artifact are provided.

Neil, M. A. A., Wilson, T. and Juskaitis, R.

A light efficient optically sectioning microscope.  Journal of Microscopy 189: 114-117 (1998).  The authors describe the theoretical and practical aspects of configuring a standard widefield microscope for reflected light structured illumination. Included is a mathematical description, microscope configuration details, and example images of a semiconductor wafer surface.

Neil, M. A. A., Squire, A., Juskaitis, R., Bastiaens, P. I. H. and Wilson, T.

Wide-field optically sectioning fluorescence microscopy with laser illumination.  Journal of Microscopy 197: 1-4 (2000).  Description of an advanced structured illumination instrument equipped with laser illumination coupled to a one-dimensional grid pattern and a rotating ground glass diffuser. The authors discuss instrument configuration and provide example images.

Barlow, A. L. and Guerin, C. J.

Quantization of widefield fluorescence images using structured illumination and image analysis software.  Microscopy Research and Technique 70: 76-84 (2007).  An investigation of the ability to acquire quantitative information from structured illumination images of fluorescent beads and cultured human cells. The authors determined that, given the proper use of image analysis algorithms, accurate information can be obtained using this technique.

Chasles, F., Dubertret, B. and Boccara, A. C.

Optimization and characterization of a structured illumination microscope.  Optics Express 15: 16130-16140 (2007).  A theoretical analysis of structured illumination in terms of sectioning strength, resolution enhancement along the optical axis, and signal-t0-noise as a function of the objective and grid parameters. Sections having a thickness of less than 400 nanometers could be obtained from a 1.4 numerical aperture objective.

Poher, V., Zhang, H. X., Kennedy, G. T., Griffin, C., Oddos, S., Gu, E., Elson, D. S., Girkin, M., French, P. M. W., Dawson, M. D. and Neil, M. A.

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode.  Optics Express 15: 11196-11206 (2007).  A report describing structured illumination microscopy using a controlled LED array in place of a grid in the objective rear focal plane. The article describes the light source and microscope configuration, as well as a discussion of structured illumination microscopy. Included are specimen images and a mathematical description of the technology.