What is a Confocal Microscope Used For?

Ordinary light microscopy cannot be used to observe a three-dimensional tissue without physically sectioning the sample. Confocal scanning microscopy, however, is an alternative observational method that provides a more direct way of seeing the three-dimensional architecture of a whole, unsectioned specimen. Electronic imaging methods allow the user to focus on a given plane in a thick specimen while rejecting light that comes from above and below the plane of focus.

Why Confocal Microscopy?

• Confocal scanning microscopy is used by researchers in biological science and other industries to obtain an image of the three-dimensional architecture of a specimen, such as a cell or tissue. Also, the procedure is used to observe the microscopic structure of a sample that for some reason cannot be sliced into sections beforehand. Even though these challenges can be met by using a complex computer-based image processing program, this method is slow and requires a great deal of computing power.

Confocal Microscopy vs. Standard Light Microscopy

• If a thick specimen is viewed with a conventional light microscope, the image obtained by focusing on any specific plane is worsened by blurry, out-of-focus readings that come from the parts of the sample lying above and below the plane of focus. For ordinary light microscopy to work well, a sample must be sliced into thin sections to be analyzed, and the section's thickness is inversely proportional to the image's clarity. However, possibly valuable information about the third dimension is lost when a sample is sectioned and, as mentioned, some samples cannot be sectioned easily at all.

How It Works

• The confocal scanning microscope focuses on a specific plane in a thick sample while rejecting light that comes from out-of-focus regions above and below that plane. This results in a clean "optical section," and from a sequence of these sections taken at various depths, a computer program can reconstruct a three-dimensional image. This strategy is fairly similar to what a CAT scanner does for radiologists investigating an entire human body, as both methods present clean sectional views of an intact structure's interior.

How It Works: The Details

• The device is generally used with fluorescence optics, and at any instant the machine's optical system focuses its spotlight onto a single point at a certain depth in the sample. An extremely bright source of pinpoint illumination is needed, and this is usually provided by a laser whose light is passed through a pinhole. A detector fitted with a pinhole aperture collects the fluorescence emitted from the illuminated sample. Only the light from the specific focus point enters the detector, and the device excludes light coming from areas outside of the plane of focus.

Specific Uses

• The confocal scanning microscope is often used to resolve the structure of various complex three-dimensional objects, such as networks of cytoskeletal fibers in the cytoplasm of cells, and the arrangements of chromosomes and genes in a cell's nucleus during different points in the cell division cycle. Pollen grains, for example, have a complex sculptured cell wall, which can only be seen clearly using confocal microscopy. The electronics industry also uses this machine and methodology to inspect semiconductors.