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Reflected-Light Optical Microscopy

Optical Imaging and Spectroscopy

  1. Jay L. Nadeau,1,
  2. Michael W. Davidson2,
  3. Richard G. Connell Jr.3

Published Online: 12 OCT 2012

DOI: 10.1002/0471266965.com057.pub2

Characterization of Materials

Characterization of Materials

How to Cite

Nadeau,, J. L., Davidson, M. W. and Connell, R. G. 2012. Reflected-Light Optical Microscopy. Characterization of Materials. 1–18.

Author Information

  1. 1

    McGill University, Montreal, Canada

  2. 2

    National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL, USA

  3. 3

    University of Florida, Gainesville, FL, USA

Publication History

  1. Published Online: 12 OCT 2012


Imaging of opaque specimens is performed using reflected rather than transmitted light. Illumination is supplied from above using orientations ranging from on-axis (brightfield imaging) to highly oblique (oblique-light microscopy; darkfield). The illuminating light might also be polarized (polarized light microscopy; differential interference contrast) or phase-advanced or -retarded (phase contrast microscopy). Each of these techniques leads to generation of contrast from different features of a specimen, and several techniques can be used to complement each other and provide information about specimen composition, feature size, height, and other properties. Reflected-light illumination is also the most common and most sensitive way to perform fluorescence microscopy on both transparent and opaque specimens. This article covers the principles behind the major techniques of reflected-light optical microscopy and gives examples of materials applications, both traditional and emerging. The set-up of the instrumentation required for each technique is given in detail, and Section “Practical Aspects of the Method” discusses the precise instrumentation and accessories needed to implement each technique. We also provide a guide to the most common imaging artifacts and pitfalls in reflected-light microscopy.


  • reflected light;
  • polarized light microscopy;
  • phase contrast microscopy;
  • differential interference contrast;
  • darkfield;
  • epifluorescence