Confocal microscopy is a very popular technique for high-quality images. It is useful when working with tissue or bio-samples due to the relatively simple sample preparation. While in a traditional microscope we have a wide field where most of the elements at the sample surface are in focus, with a confocal microscope, a sample can be observed at different depths using spatial filters that eliminate out-of-focus light.
Confocal microscopy was invented in 1955: a sample is scanned and a point-to-point image is created. Light passes through a series of pin holes that eliminates unfocused light thus increasing image resolution. We discuss a similar system here: the scanning of the sample can be made either by moving the sample or by xy scanning mirrors. Figure 1 shows a schematic of the system.
Figure 1. Fluorescent Microscope. By derivative work: Henry Mühlpfordt (talk)
In terms of optical components, we require a very intense light source. After the light source, we have a filter (usually referred to as an excitation filter), where only a very specific wavelength passes through. From this filter, we go to a dichroic mirror. Dichroic mirrors have a very specific transmission and reflection coefficient for two different wavelengths, that is they allow one wavelength to be transmitted while reflecting the other wavelength. In our case. we will reflect the light coming from the excitation filter, and this beam will be focused by a lens into a sample. The sample is then excited by this specific wavelength and fluorescence takes place in the biomolecule we are trying to detect. The light emitted by the biomolecule due to fluorescence is then transmitted back to the dichroic mirror. On this second pass, the emitted light passes through the mirror and then through a pinhole (this one called emission filter) and then to the ocular or camera.
One of the great advantages of confocal microscopy is the ability to create 3D sample images. Data obtained from optical slices along the optical axis are used to reconstruct a 3D image using software and image processing. Fig. 2 shows a 3D reconstruction from slices of a suspension of 2 mm diameter PMMA particles.
Figure 2. Three-dimensional reconstruction of a series of 2D images of PMMA spheres suspended in solution. Image from Denis Semwogerere and Eric R. Weeks
A confocal microscope provides a significant imaging improvement over conventional microscopes. It creates sharper, more detailed 2D images, and allows collection of data in three dimensions.