Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. The key feature of confocal microscopy is its ability to produce blur-free images of thick specimens at various depths. The principle for this special kind of microscopy was developed by Marvin Minsky in 1953, but it took another thirty years and the development of lasers as near-ideal point light sources for confocal microscopy to become a standard technique toward the end of the 1980s.

Image Formation
In a laser scanning confocal microscope a laser beam passes a light source aperture and then is focused by an objective lens into a small (ideally diffraction-limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole the fluorescent light is detected by a photo-detection device (photomultiplier tube (PMT) or avalanche photodiode) transforming the light signal into an electrical one which is recorded by a computer.

As seen in the figure the detector aperture obstructs the so called out-of-focus (or out-of-plane) light: fluorescent light not originating from the focal plane of the objective lens. Light rays from below the focal plane come to a focus before reaching the detector pinhole, and then they expand out so that most of the rays are physically blocked from reaching the detector by the pinhole. In the same way, light from above the focal plane is focused behind the detector pinhole, so that most of this light also hits the edges of the pinhole and is not detected. However, all the light from the focal plane (solid lines) is focused at the pinhole and passed to the detector. In this way, out-of-focus information from above and below the focal plane is greatly reduced, which results in sharper images compared to conventional microscopy techniques. The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane using one or more (servo-controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied as slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.