Confocal Optical Section Thickness

The image of a point (spot) that is created by an optical system is called the “point spread function” (PSF). The point spread function describes the distribution in three dimensions of light originating from a dimension-less spot. The core of this diffraction pattern has an ellipsoid shape, and that controls the optical resolution. The radial dimension rules the lateral resolution; the axial dimension rules the depth of focus and thus the slicing performance.

For confocal sectioning, the goal is to transmit only the inner core of the PSF, which defines the optical section. As a matter of fact, the pinhole diameter controls how much light from outside the core is transmitted. The optical section that is in the range of the size of the diffraction pattern is thus obviously not sharp-edged like a slice of bread, but is characterized by a comparably flattish slope. The distance in z that connects the 50 % intensities on both sides of an intensity profile is called the “full width half maximum” (FWHM), and by convention this is used as a measure of the thickness of optical sections. The optical sectioning performance is typically measured by focusing through a surface mirror that models an infinitely thin structure in z. This method is (comparably) easy and is used to examine the performance of confocal microscope systems. Alternatively, and more realistically, z sectioning performance is measured with fluorochromated latex beads (see Figure 1). The fluorescent beads allow the performance to be measured in non-coherent conditions, which is true for fluorescence imaging and thus fits the vast majority of confocal applications in biomedical sciences. The diffraction-limited optical sections in reflected light mode (mirror) are significantly thinner as compared to sections in fluorescence mode. This is important to keep in mind if comparing figures from literature.

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