"Confocal Microscopy" is short for "Confocal Laser Scanning Microscopy" (CLSM). This is a form of microscopy that uses a focused laser beam to excite fluorescence in a specimen. The fluorescence produced by the interaction of the laser with the labeled parts of the specimen is then detected by highly sensitive electronics and the signal is used by a computer to generate high resolution image sets called image "stacks". Once collected, the stacks can be reconstructed to make highly detailed, accurate, three-dimensional renderings.

Typical CLSM equipment. This system is optimized for work with live samples and uses an inverted microscope with objective lenses beneath the stage. The inverted design allows the observing of live specimens growing in Petri dishes.

An image stack. This layer of cells was optically sectioned by a confocal microscope. The stack was collected in 0.25 micron steps. It is represented here in a way that makes it easy to see that different events are occurring at different levels within the cell layer. Not all optical sections are shown.

Confocal microscopy is an evolutionary development that grew out of an older fluorescence-based technique called epifluorescence microscopy. For a description of what epifluorescence microscopy is and how it works, click here:

Why "con" and "focal"? This is shorthand for "conjugate focal plane". In a nutshell, as a specimen fluoresces,  the emitted fluorescence is brought back to a second focus - the "conjugate focus". This is the innovation that makes the confocal microscope able to optically section through thick or living specimens with great clarity and minimum blur. See the image below for an example.

The excitation source and dichroic mirror have been left out of this diagram for the sake of clarity. Starting at the bottom, fluorescence is emitted from the specimen from all fluorophore-labeled structures excited by the laser. Three type of ray are generated and focused by the objective lens: 1) a ray from structures below the plane of focus (red), 2) a ray from structures at the plane of focus (black), 3) a ray from structures above the plane of focus (blue). The three rays are brought back to focus at the plane of the iris (the conjugate focus). Only the ray from the in-focus plane passes through the iris. The out-of-focus rays are blocked and therefore the out-of-focus blur is elminated.

A more detailed description of CLSM follows.

In the CLSM, a laser beam delivered to an x-y deflection mechanism, passes through the objective lens of the microscope and is rasterized across a fluorescently labeled specimen. The emitted fluorescence passes through the same objective lens, passes through a series of filters that allow detection of chosen wavelengths only, passes through a confocal aperture, and is finally detected by a photodetector (photomultiplier tube).

The photomultiplier converts the detected fluorescent light into a proportional electrical signal that represents the level of fluorescence at the point from which the laser generated the fluorescence. The number of points where the fluorescence is measured across a specimen is know as the digital sampling density or, more commonly as sampling resolution. Each point becomes a pixel in the final image. Therefore, a specimen sampled with a density of 512 points in the x direction, and with 512 lines rastered in the y direction creates an image that is 512 pixels by 512 pixels.

The popularity of the confocal microscope is due to its' unique ability to produce clear, blur-free optical sections of thick and/or living samples. This ability to nondestructively section a specimen comes from passing the emitted fluorescent light through the confocal aperture. The aperture is located at a point within the scanning mechanism where the emitted fluorescence is brought to a second point of focus. This second point is known as the conjugate focus - which is how the term "con - focal" arrived. In essence, the aperture serves as a spatial filter by eliminating out-of-focus light originating above and below the focal point. Thus, any fluorescence detected by the photomultiplier tube has passed through a "spatial filter" (the aperture), while the out of focus rays are blocked by it. Refer to the diagram above for a simplified physical arrangement.