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 tutorial initializes with the Spinning Disk rotating at a moderate speed and a light beam passing through pinholes on the left-hand side of the disk. Both the number of pinholes and the disk speed have been modified (reduced number, but larger pinholes and slow disk speed) for purposes of the tutorial. In order to operate the tutorial, click on the Zoom In button to show an enlarged view of the disk area illuminated by the incoming light beam. The specimen (a single nucleus) is also illustrated in this window with the beam sweeping across. Use the Rotation Speed slider to increase or decrease the disk speed and note changes to the illumination pattern superimposed on the specimen.
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.
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.