Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Optical Sectioning

Optical Sectioning with Structured Illumination


The goal of structured illumination technology (including the ZEISS ApoTome) is to improve the images of thick specimens using a combination of optical manipulations coupled to computational algorithms. Through a remarkably simple process, the blurring produced by defocus can be converted into an effective tool for separating light in the focal plane from obstructing light produced in remote areas of the specimen that are far removed from focus. Among the numerous advantages of structured illumination is the ability to produce crisp optical sections having a thickness that coincides with the objective resolution. This article explores optical sectioning with the ZEISS ApoTome.

Even through the image sequences produced by laser scanning confocal microscopy, widefield fluorescence deconvolution, and structured illumination techniques are often referred to as optical sections, they differ significantly from true physical sections in that their top and bottom edges are not sharply defined. In a traditional physical tissue section that has been cut by a knife in a microtome, there is no ambiguity about which region of the specimen contains each point in the image. A specific point in the tissue was either included in a particular section or it was not, but there is no intermediate state. An optical section, however, includes some locations that are faithfully reproduced (in effect, present at their true intensity), and other locations above and below that are reproduced at less than their true intensity (particularly at the edges). There exists no sharp cutoff that demarcates what is included in the optical section and what is excluded. Instead, there is a continuous decrease in the ratio of image to specimen intensity for locations that reside further away from the mid-point of the section.

Contributing Authors

Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.