Confocal microscopy is used for three dimensional imaging of fluorescence in biological tissue. Below are sections of different spectral channels from deep within an engineered tissue. Confocal microscopy achieves this by capturing florescent light originating only from the focal point of the objective. The focus may then be rasterized through the sample to create a 2D image or a 3D volume.

To understand how this is achieved, see the figure below. Starting at the source, a point source of light (colored blue in the diagram) is reflected off a long-pass dichroic mirror and focused through a microscope objective into the sample. The light then causes certain molecules in the sample to fluoresce, or re-radiate the incident light at a longer wavelength (red lines in diagram). Some of the fluorescent light is radiated in the epi-direction, and focused back through the objective. Since the wavelength of the fluorescent light is longer than that of the source light, the fluorescent light passes through the long-pass dichroic mirror, instead of being reflected. The fluorescent light (solid red line in diagram) must then pass through a pinhole, placed at the objective's conjugate focal plane, in order to be detected by the photodetector. Fluorescent light that originates from outside the focus of the objective will not be in focus at the conjugate focal plane, and hence will be rejected by the pinhole (dashed red lines in diagram).