Confocal Microscopy - Basic Concepts

Fluorescence microscopy is a wonderful tool, especially combined with specific antibodies or fluorescent protein constructs, but it does have its limitations. One of the biggest problems with fluorescence microscopy is that samples thicker than a few microns are very hard to focus. This is because light from above and below the focal plane is also visible and it interferes with the in-focus light. One way to get around this is to cut the sample into very thin sections, so that the all the visible light is coming from the focal plane. Another solution is to take optical sections. This is where the sample is left intact but the out-of-focus light is eliminated. This approach is employed in confocal microscopy.

First of all, the sample needs to be made visible with the use of fluorophores, or fluorescent tags. These are molecules that can be excited by a specific wavelength of light and then emit light at a different, longer, wavelength. For examples of some different fluorophores and their excitation and emission spectra, have a look at this online tool from Molecular Probes.

Very specific wavelengths of light can be used to excite the fluorophore by using a laser. This means that you will only see fluorescence from molecules that are excited by that wavelength, and not others that are excited by other similar wavelengths. The laser is also highly focused, so less of the sample is being bathed in light and emitting fluorescence. Both of these help to eliminate the amount of interfering fluorescence. To reduce the glare from unfocussed light even more, confocals have a pinhole, which restricts the light from the sample that reaches the detector. The smaller the pinhole is, the less light gets through and the more precise is the focal plane. A larger pinhole provides a brighter image but it is gathered from thicker portion of the sample, which means some of it will be out of focus.

By collecting a series of optical sections, the sample can be reconstructed in 3D - this is called a projection. A projection can be used to show the full depth of the sample in clear focus, and it can be rotated to show the spatial relationship between points that sit on top of one another when viewed from the top.

More Info on:

Bleaching

DIC

Fluorophores

GFP

Immunolabelling

Spectral Imaging

Imagine you are looking at a hollow fluorescent ball.

With normal fluorescence microscopy, out-of-focus as well as in-focus light will be visible, giving the image a blurred appearance.

By taking optical sections through the ball, you can collect in-focus images of ball from top to bottom.