In a confocal imaging system a single point of excitation light (from the first pinhole A) is focused onto a confocal spot (S) in the specimen. With only a single point illuminated, the illumination intensity rapidly falls off above and below the plane of focus as the beam converges and diverges, thus reducing excitation of fluorescence for interfering objects situated out of the focal plane being examined. Fluorescent light (i.e. signal) passes back through the dichroic reflector and then passes through the second (exit) pinhole (B), which is confocal with S and A. The exit pinhole can be made small enough that any light emanating from regions away from the vicinity of the illuminated point will be blocked by the aperture, thus providing yet further attenuation of out-of focus interference. A photomultiplier detector (PMT) provides a signal of the light passing scanned S1, S2, S3, S4, etc.(not shown), as the specimen is scanned. A computer is used to control the sequential scanning of the sample and to assemble the image for display.
In summary, a confocal imaging system achieves out-of-focus rejection by two strategies: a) by illuminating a single point of the specimen at any one time with a focused beam, so that illumination intensity drops off rapidly above and below the plane of focus; b) by the use of blocking a pinhole aperture in a conjugate focal plane to the specimen so that light emitted away from the point in the specimen being illuminated is blocked from reaching the detector.