Axial resolution in spinning disk microscopy is largely defined by the size of the pinhole or slit and the separation distances between these apertures. In cases where pinholes are too large or placed too close together, fluorescence emission originating in regions removed from the focal plane can pass through adjacent pinholes and obscure specimen detail and reduce resolution. This interactive tutorial explores the effects of pinhole crosstalk with a simple model that is based on a virtual spinning disk placed directly in the image conjugate plane.
The tutorial initializes with a diagram of light passing through the pinhole of a spinning disk situated in the specimen plane. Light emerging from the pinhole fills the objective front lens element. Adjacent to the objective diagram is a Specimen window that illustrates effects of relocating the spinning disk on the specimen image. In order to operate the tutorial, use the Focal Plane slider to move the spinning disk away from the specimen plane. As the disk moves farther away, the emission light diminishes and the image loses confocality. New specimens can be selected using the Specimen pull-down menu.
Illustrated in Figure 1 is the effect of light originating from remote focal planes passing through adjacent pinholes to create a haze level that obscures the sharp specimen detail normally observed in confocal microscopy. In Figure 1(a), a portion of the red fluorescence emission arising from a theoretical specimen point reaches an objective front lens having an acceptance half-angle of 45 degrees (equal to a numerical aperture of 1.0). Fluorescence is emitted in all directions from the specimen point, which is presented as being located in the center of a pinhole in a Nipkow disk, but the diagram is restricted to show only that emission actually entering the objective. In reality, the Nipkow disk is located in the intermediate image plane (on the other side of the objective). However, the masking effect of the disk can be observed in any of the conjugate image planes, including the focal plane of the specimen, as illustrated in Figure 1. Thus, for the instructive purposes of this illustration, the pinhole is shown sampling the light near the focal plane.
The Nipkow disk pinhole diameter in Figure 1(a) is assumed to be a single Airy pattern unit in diameter with reference to the focal plane (in effect, approximately 0.5 micrometers). It is also assumed that essentially all of the fluorescence emission representing the central maximum of the Airy disk represented by the point object proceeds through the pinhole and towards the objective. A view of the Nipkow disk from the side opposite the objective is presented in Figure 1(b), and the pinhole diameter (D; 0.5 micrometers) and inter-pinhole spacing (S; 2.5 micrometers) are indicated on the drawing. The total light transmission through a disk having a D/S ratio of 1/5 is approximately 4 percent, consistent with typical spinning disk microscopes that are not equipped with microlens arrays. Relocating the specimen point approximately 1 micrometer beneath the focal plane (Figure 1(c)) reduces the amount of light passing through the pinhole due to the fact that much of the light emanating from the point now strikes the bottom of the disk (Figure 1(d)) and is reflected from the surface.
Relocating the specimen point to a distance equal to S (2.5 micrometers) away from the focal plane enables some of the emission light to pass through the first ring of neighboring pinholes (Figures 1(e) and 1(f)). As a result, more fluorescence emission now passes through the six peripheral pinholes than through the central pinhole, mimicking the background signal haze for a highly fluorescent point source positioned away from the focal plane in a thick specimen. The situation is similar for slit disks, except that a larger fraction of the light will pass through the adjacent slits than the neighboring pinholes. Note that when the specimen point is positioned at the location in Figure 1(e), emission light is now spread over a much larger diameter on the Nipkow disk and the excitation is likewise diminished. As the specimen point is lowered still farther away from the focal plane, the number of photons passing through the disk continues to diminish until some of the emission light begins to pass through the ring of secondary neighbors when the point is approximately 5 micrometers beneath the disk.
In summary, calculations involving the axial resolution of spinning disk confocal microscopes must take into account a variety of specimen parameters in addition to the physical configuration of the microscope. The distance between pinholes or slits can be increased to improve the axial resolution at the cost of signal, but the overall performance of a well-designed spinning disk microscope is determined to a greater extent by the efficiency of light transmission through the disk than whether the apertures are slits or pinholes. Critical specimen variables include staining patterns, thickness, and orientation. For those specimens where stained structures are confined to a thin layer, or where the stain is concentrated into small, but sparse regions, spinning disk microscopes can approach the axial resolution performance of laser scanning confocal instruments. However, heavily stained, thick specimens invariably will perform more poorly in spinning disk microscopes and this factor should be taken into account when planning investigations.
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.