Confocal microscope

A confocal microscope combines focused illumination with a spatially restricted detection path aligned to the same specimen location. In a scanning implementation, a laser focus moves across the specimen and a pinhole in a conjugate image plane rejects much of the emitted light originating away from focus.

A photodetector records the signal at each scan position to build the image. Repeating at different depths gives confocal optical sectioning. Closing the pinhole improves rejection but discards useful photons, so signal-to-noise ratio and section thickness trade against one another. Measuring a small fluorescent bead through focus tests the actual three-dimensional response rather than assuming the nominal pinhole setting fixes it.^[ref]