Confocal microscopy has become a vital tool for the study of neurogenesis. The goal of this page is to explain how confocal microscopy works, and show that when combined with other techniques, why confocal microscopy is crucial to the study of neurogenesis.
Invented in 1955 by Marvin Minsky but more recently developed and commonly used confocal microscopy uses point by point illumination of a specimen and collection of the returning rays by a photomultiplier (a sensitive detector of light). This process constructs images of the specimen on a long-persistence screen. By illuminating a single point of the specimen as well as using a pinhole to collect only rays of light reflected from the focal point, confocal microscopy eliminates unwanted rays of light thus increasing resolution.
To create an image of the entire specimen, current methods use motor driven mirrors that focus light on vertically and horizontally scanning mirrors. These mirrors then focus the light on different parts of the specimen. In both techniques, the light, either reflected or given off by the specimen, is then sent through a filter with a pinhole size opening that collects these rays from the focal point of the mirrors.
Fluorescence Confocal Microscopy
The most common of modern techniques utilizes fluorescent dies that emit rays of light when light of a shorter wavelength is absorbed. If the observer shines an excitation light on the dyed specimen, the fluorescent molecule (fluorophore) may absorb photons and increase its excitement state. At this point, the molecule may undergo spontaneous emission of light due to a need to return to ground state. This emission will be at a longer wavelength making the specimen clearly visible. Fluorescent microscopy uses a dichroic mirror to reflect light to the specimen. This dichroic mirror allows light of a longer wavelength to pass back through it to the eyepiece. This allows the viewer to see the specimen in a different color from the excitation light. The one systematical problem of confocal microscopy is diffraction of light on the optical and lateral axis causing limitations in resolution.
Advantages of fluorescence microscopy in studying neurogenesis include:
- Increased sensitivity due an increase in signal to noise of technique
- Desired structures can be isolated using certain fluorophores that are bound to antibodies which specifically bind to certain molecules, for example immunohistochemical markers (see also immunohistochemistry)
- Gives the ability to use multiple fluorophores. When excitation light is changed, the different fluorophores highlighting different structures of the specimen can be viewed.
Confocal Microscopy Images of Neurons: