Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Optical Sectioning

Optical Sectioning Microscopy

Among the most critical artifacts to consider in widefield fluorescence microscopy arises from the fact that regardless of the focal point, illumination from the objective produces fluorescence throughout the entire specimen volume. As a result, widefield fluorescence images often contain a high level of background signal that obscures specimen detail and dramatically reduces contrast. Confocal microscopy largely eliminates the background signal problem by incorporating a pinhole near the detector that resides in a plane conjugate with the focus point. In widefield microscopy, optical sections can be obtained using either computational (deconvolution) or structured illumination techniques. This interactive tutorial explores the basic concept of optical sectioning using an animated cell model.

New techniques developed for widefield fluorescence microscopy are centered on non-uniform illumination methodology. The most successful of these approaches are aperture-correlation and sine-modulated illumination microscopy. In aperture-correlation microscopy, a large number of images must be gathered at each focal plane, which ultimately results in reduced signal (due to photobleaching) and potential specimen damage. In contrast, the sine-modulated techniques, such as structured illumination, employ a sinusoidal illumination pattern that is focused by the objective onto the focal plane of the specimen. At least three sequential images are gathered at each focal plane by laterally shifting the pattern by one-third. Each of the resulting images contains both in-focus and out-of-focus information. However, the in-focal information is modulated by the sinusoidal illumination pattern, whereas the background (out-of-focus) signal is affected far less. A final image can be generated using algorithms that remove features common to the images, thus resulting in a crisp and sharply focused optical 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.