Reflected‐Light Optical Microscopy

Abstract 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.