It is similar to wide-field fluorescence microscopy in that fluorescence optics are used. Unlike traditional fluorescence microscopes, however, confocal microscopy focuses a laser light source onto a defined spot at a specific depth within the sample. This creates the emission of fluorescent light at the exact point defined and, using a pinhole aperture inside the optical pathway, out-of-focus light is removed from the field of view. This leads to much clearer images being produced.

The term Confocal is derived from the conjunction of two focal planes: the objective lens focus point and the focus point where the aperture is placed.

Confocal imaging increases resolution and contrast through the use of the pinhole spatial filters. One pinhole is placed in a conjugate plane with a scanning point on the specimen, and a second pinhole is positioned in front of the detector (e.g., photomultiplier tube).

This effectively removes out-of-focus light and provides a crisper image with the maximal resolution possible for the objective lens being used.

Modern confocal microscopes offer high-power, high-resolution, and multicolor capabilities. They are commonly used to collect high-quality multispectral images of biological and non-biological samples across the visible range. Their maximum magnification level, in general, is 1000x when combining a 10x ocular with a 100x objective lens.

In addition, specialized confocal microscopes can image living cells, and work at very high speeds to perform live-cell imaging without causing severe photo damage, photobleaching, or phototoxicity. Confocal microscopes are even used to assist with in vivo preparations.

Furthermore, modern units are often designed as completely integrated confocal systems. In these systems, an optical microscope works together with a detector or multiple detectors, a computer, multiple laser systems, a wavelength selection device, and beam scanning assembly to produce images and obtain valuable information about a sample. These integrated systems are typically referred to as digital or video imaging systems.

Basic confocal microscopes and more robust systems are used extensively in the life sciences (e.g., biology and cell biology), materials science, and semiconductor inspection. When acquiring one, it’s essential to understand your needs, as this will inform whether or not you’ll want to lease a fully integrated system or something a little less complex.