Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Optical Sectioning

Spinning Disk Fundamentals

Explore how light passes through the pinholes on a spinning disk microscope to produce multiple excitation beams that are swept across the specimen as the disk spins. The Nipkow disk (termed Spinning Disk in the tutorial window) is located in a conjugate image plane and a partial rotation of the disk scans the specimen with approximately 1000 individual light beams that can traverse the entire image plane in less than a millisecond to scan the specimen in parallel.


The Nipkow disk, regardless of whether it contains multiple sets of spirally arranged pinholes or parallel slits, is always positioned in one of the conjugate image planes in a spinning disk confocal microscope. Pinholes or slits are illuminated from the rear of the disk (the side opposite the objective pupil) so that their vastly reduced images are focused by the objective onto the specimen plane. As the Nipkow disk rotates, the specimen is raster scanned by successive sets of miniature pinhole images or so-called "virtual" pinholes in the detector conjugate plane produced by orthogonally arranged slits. The light emitted or reflected from each illuminated point on the specimen is once again focused by the objective onto a corresponding pinhole on the Nipkow disk. Depending upon the microscope design, the exit pinhole may be the same pinhole that provided the scanning spot or it may be a pinhole that is located on the opposite side of the disk. In all Nipkow disk systems, a relatively large separation distance between adjacent pinholes (relative to their diameters) must be maintained in order to minimize crosstalk from fluorescence emission or reflected light. This pinhole size and spacing issue is always a tradeoff between generating enough light to successfully image the specimen and maintaining a high degree of confocality.

Contributing Authors

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