Confocal microscopes use a pinhole to reject out-of-focus light, allowing for high resolution fluorescent images. Moving mirrors scan light across a sample, creating optical slices that can be combined to create a 3D image. They are commonly used in life science labs but require a dedicated dark room.
A confocal microscope is a special type of fluorescence microscope used to obtain high quality 2D and 3D images. With a basic (wide field) fluorescent microscope, light of a particular wavelength is reflected off a sample, and the sample absorbs some of that light and reflects the rest at a longer wavelength. The longer wavelength light passes through a dichroic mirror, which allows the longer wavelength light to pass, but not the original (shorter) wavelength light. The person looking at the sample sees only the light that has passed through the dichroic mirror. Basic fluorescent microscopes capture both in-focus and out-of-focus light, which works well for large specimens where very high resolution isn’t required. It doesn’t work so well for viewing fine details. A confocal microscope is required to acquire high resolution fluorescent images.
The confocal microscope uses a pinhole to reject out-of-focus light. Instead of light only passing through a dichroic mirror, light passes through both a dichroic mirror and a pinhole, meaning that the person looking at the sample only sees a single point in the sample. To view an entire sample, moving mirrors scan light across a rectangular plane of the sample, and a computer collects the light emitted as the sample is scanned. A full scan will result in an optical slice, or cross section, of the sample. This type of microscopy is called confocal laser scanning microscopy, in part because a laser is used as the light source.
To create a 3D image, several scans are taken, creating several “slices” of the sample, and a computer combines each slice to create a three-dimensional image. The microscope can be programmed to vary the space between the slices, depending on how sharp the 3D image the user wants to get. The microscope can also vary how fast it scans an image, with slow scanning providing greater resolution than fast scanning.
The confocal microscope has become a very commonly used instrument in many life science laboratories. They are expensive, and a bit large, and often have rooms dedicated to their use, because the room has to be dark to get a good fluorescent picture. Although confocal microscopes are most commonly used to view cells and structures within cells, they can be used to obtain an image of any three-dimensional fluorescent sample and are sometimes used to view semiconductor materials.
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