The amount of light transmitted through the Nipkow disk in spinning disk microscopy is determined by the diameter of the pinhole or slit and the distance between these apertures. Transmission through a pinhole disk averages approximately 4 percent of the incident light when the pinhole aperture is 50 micrometers in diameter and pinholes are separated by a 250-micrometer interval. This tutorial explores how the amount of light passed through a disk can be increased by using microlens arrays on the upper disk in a two-disk system.
The tutorial initializes with diagrams of incident light passing through a microlens array on a two-disk Yokogawa scanning system. Light gathered by the microlenses on the upper disk is passed to a pinhole array on the lower disk, with approximately 58 percent of the light being passed through the system. In order to operate the tutorial, use the Microlens Size slider to increase or decrease the amount of light gathered by the microlens elements. Turning the Microlens Button to the OFF position removes the upper disk, demonstrating the lower transmission of the lower disk without the assistance of the microlens array.
In order to obtain the confocal effect in spinning disk microscopy, it is necessary to strategically place the pinholes far enough apart in a geometrical pattern that avoids emission light returned by the specimen from entering the wrong pinhole. Slit apertures, rather than pinholes, can be used to increase the transmitted light budget, but these disks are also sensitive to unintended spillover of emission into adjacent slits. Furthermore, slit scanning will result in a slight decrease in lateral resolution along the long axis of the slits (roughly by a factor of 1.2 to 1.4). Slits can be arranged as linear arrays (oriented both parallel and perpendicular) or as spirals. The major trade-off between slits and pinholes is that slits provide brighter illumination and higher signal throughput, whereas pinholes generally attain higher axial resolution. The fill factor, defined as the percentage transmission (T) through a Nipkow-style disk, is higher for slit disks than for pinhole disks when the ratio of the diameter to the distance between pinholes or slits is fixed according to the equations:
TPinhole = (D/S)2 × 1000 (1) TSlit = (D/S) × 1000 (2)
where D is the diameter of the pinhole or slit and S is the distance between pinholes or slits. To demonstrate relative light transmission capabilities between the two geometries, consider the case for pinhole and slit apertures having a 50-micrometer diameter (or width) and a 250-micrometer spacing. In this configuration, pinholes will transmit 4 percent of incident light, whereas slits will transmit five times that amount, or 20 percent. The transmission through a pinhole disk can be increased by using a smaller spacing interval between pinholes, but even reducing the spacing between adjacent pinholes in half (to 125 micrometers) only increases the transmission to 16 percent. Furthermore, decreasing the spacing distance (S) between pinholes (or slits) results in more emission light being able to return through neighboring pinholes, decreasing the axial resolution. Increasing the slit or pinhole diameter (D) also leads to a reduction in axial resolution.
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.