Confocal microscopy allows the recording of optical sections. This is made possible by a variable aperture located in the conjugated focal plane (con-focal plane) on the optical axis: the confocal detection pinhole. It blocks out-of-focus light from reaching the detector, leading to a resolution improvement along the optical axis (Z-direction) by a factor of approximately 1.4 compared to widefield microscopy (for more information on the optical path of a confocal microscope please refer to the Leica Science Lab). In the confocal plane each point in the sample appears as a typical diffraction pattern also known as the Airy pattern (Figure 1), with the central diffraction maximum referred to as the Airy disc. The out-of-focus light is represented as the higher-order maxima of the pattern. Typically the pinhole is adjusted such as to permit the principal maximum, the in-focus light, to the detector for recording. This setting is referred to as 1 Airy unit.
Since the addition of the pinhole on the other hand leads to light loss it is desirable to have a pinhole with adjustable size to balance resolution gain and light loss (for details on resolution and Airy units please refer to Leica Science Lab). An adjustable pinhole diameter is technically difficult to realize for a perfectly circular geometry, which is why other geometries are preferred. The most common shape is the hexagonal pinhole geometry, similar to the apertures in objectives used in photography. At the confocal plane light has not yet been detected, but it must pass to the detection system, where it is split up along the spectral dimension for multispectral imaging. After light dispersion, by a prism in Leica confocals, each color gives rise to a unique diffraction pattern (Figure 2). The better their principal maxima are separated the better the spectral resolving power of the confocal microscope. The pinhole geometry has a strong impact on the diffraction pattern (Figure 3, top row). It turns out that a square pinhole arrangement is most beneficial for cancelling the problematic secondary maxima and hence results in improved spectral resolution (Figure 3, bottom row). In fluorescence microscopy each (biological) structure of interest is labeled specifically with a unique color and their colocalization conveys important information about biological function. The spectral specificity by means of a square pinhole therefore can result in improved functional characterization of the multicolor sample.
Figure 1: The confocal pinhole induces a diffraction pattern in the confocal plane. One Airy unit is defined as its principal (zero-order) maximum and is the physical equivalent of the in-focus light. By changing the pinhole size one can balance optical resolution and light intensity. The physical size of the pinhole depends on the exact geometry of the beam path and the magnification of the optics. Therefore Airy units are more useful for comparing pinhole sizes than its physical diameter.
Figure 2: Diffraction patterns for two colors after passing through a prism. In multi-spectral imaging spectral specificity is influenced by the ability to resolve two adjacent diffraction patterns.
Figure 3: Diffraction patterns caused by different pinhole geometries (schematical illustration based on computed diffraction patterns). Top row: Circular (A), hexagonal (B) and square (C) pinhole. Bottom row: Separation of two adjacent patterns, such as caused by different colors for circular (D), hexagonal (E) and square (F) geometries. Color separation is important for multispectral imaging. Thanks to its lack of intensity between the two first-order diffraction maxima the square pinhole geometry allows for the easiest suppression of higher orders. The gain in spectral separation is approximately 1.5 (US patent 6,801,359 B1).