Confocal microscope

A confocal microscope is a light microscope designed to produce images with a very small depth of field, called optical sections.

Confocal microscope technique

When using a conventional light microscope, if an object is thick, a clear image of a part of the object can only be obtained within the focal plane. The focal planes located beneath or above make the image blurred.

The principle of the confocal microscope is that it removes light from upper and lower optical sections using pinholes positioned at the light source in front of the detector. To do this, a diaphragm is placed in front of the detector in the focal plane and conjugated to the focal plane of the lens (hence the description "confocal"). Therefore, only light from the focal plane reaches the detector.

The light source is a laser that scans the object being examined point by point. It uses a semi-reflective mirror in its "reflection" configuration that reflects the ray from the object towards a detector. The detector can then measure the light intensity at each point and store it in the computer.

Once the object has been scanned along the X and Y axes and a two-dimensional image has been obtained, the surface containing the object is moved by an increment, dz, and the scan is repeated. "Sections" are then memorised which can then be processed by computer to produce three-dimensional images of the object.

Use of the confocal microscope

Resolution is better than a conventional light microscope (180 nanometres width and 400 to 600 nanometres depth). The images are therefore much clearer.

Confocal microscopy is extensively used in molecular biology to examine the interaction between two proteins (FRET) or the subcellular location of certain proteins.