3.3E: Confocal Micropscopy
- Compare and contrast confocal and fluorescence microscopy
Confocal microscopy is a non-invasive fluorescent imaging technique that uses lasers of various colors to scan across a specimen with the aid of scanning mirrors. The point of illumination is brought to focus in the specimen by the objective lens. The scanning process uses a device that is under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube through a pinhole. The output is built into an image and transferred onto a digital computer screen for further analysis. The technique employs optical sectioning to take serial slices of the image. The slices are then stacked (Z-stack) to reconstruct the three-dimensional image of the biological sample. Optical sectioning is useful in determining cellular localization of targets. The biological sample to be studied is stained with antibodies chemically bound to fluorescent dyes similar to the method employed in fluorescence microscopy. Unlike in conventional fluorescence microscopy where the fluorescence is emitted along the entire illuminated cone creating a hazy image, in confocal microscopy the pinhole is added to allow passing of light that comes from a specific focal point on the sample and not the other. The light detected creates an image that is in focus with the original sample. Confocal microscopy has multiple applications in microbiology such as the study of biofilms and antibiotic-resistant strains of bacteria. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.
Key Points
- Confocal microscopy requires immunoflurescence staining of biological samples.
- Confocal microscopy serves to control depth of field, eliminate background, and collect optical sections.
- The use of confocal microscopy has expanded to study both fixed and live cells with the ability to quantify targets.
Key Terms
- photomultiplier tube : A vacuum tube that detects ultraviolet, visible, and near infrared light and multiplies it 100 million times.