Functions, Uses and Advantages of the Confocal Scanning Laser Microscope
The confocal laser scanning microscope or CLSM is one of the most advanced types of microscopy available today. The confocal scanning laser microscope was developed out of the technology that produced the fluorescence microscope. This type of microscopy relies on fluorescence emission to produce images, thus, allowing the observer to view specific components, structures and processes in living tissues and cells. The confocal laser scanning microscope is used in many areas of medical, biological and live sciences.
The principle behind the technique
In the more conventional type of fluorescence microscopy, ultraviolet light sourced from lamps made of tungsten or xenon is used to excite the atoms within a specimen. This in turn produces the fluorescence that is viewed through the microscope. With the confocal scanning laser microscope, a laser beam is used. The objective lens focuses the laser beam to pass through a specific area of the specimen. Usually, this area is diffraction limited to allow for a more effective processing of light.
Once the laser has passed through, the specimen produces both its own fluorescent light and the reflected light from the laser. These two beams of light are then collected by the objective lens and with a beam splitter in the microscope, separated once again. The laser light passes through while the fluorescent light is reflected onto a detection apparatus. This light is passed further through a pinhole and is detected using a photomultiplier tube, an avalanche photodiode or other such photodetection device. This light is transformed to an electrical pulse that a computer can read.
The detector device is also responsible for blocking any light that is not sourced through the focal point, because this light is out of focus. The result is a very sharp image of the specimen, even sharper than the ones produced by epifluorescence microscopy, a popular fluorescence technique highly favored by many users.
The laser beam used in this type of microscopy is capable of producing an image of the specimen in pixels, which, when translated by the computer as a whole image, can vary in intensity depending on how much of the fluorescent light is detected. Since scanning speed can vary, the image clarity and sharpness can also change, with slower speeds producing better pictures and a more efficient signal to noise ratio. By raising and lowering the specimen stage, the microscope can even be used to produce an image of the specimen in 3D.
Advantages of confocal laser scanning microscope
Generally, the problem with fluorescing specimens is that they can produce light that sometimes interferes with the microscope’s ability to produce high resolution pictures. With the confocal laser scanning microscope, this is not a concern.
In terms of image quality, the confocal scanning microscope can also offer excellent resolution. The laser used in scanning the specimen is capable of sending varying degrees of fluorescence in specific areas and the instrument itself is capable of detecting these minute discrepancies, no matter how discrete. Other microscopy techniques like wide field illumination, for example, have a limited capability of producing high resolution images.
Another very useful advantage of the confocal laser scanning microscope is its ability to provide a closer inspection of the specimen in a direct yet non-invasive way. Because the specimen can be manipulated and the instrument adjusted accordingly, optional section involving living specimens may be performed. This allows the specimen to produce a series of optical sections using thick specimens. Living specimen samples may also be examined intact, with little preparation required. Dyes and stains used to encourage fluorescence in the specimens can also be minimized, allowing the specimens to survive longer and be observed live.
Depth of field, usually a challenge in other types of optical microscopy, is relatively controllable using the confocal laser scanning microscope. Any degradation involving out of focus imaging is also eliminated.
Another useful advantage of the confocal laser scanning microscope is that it is designed to be easy to set up and operate. Later models have also evolved to allow multiple users.
Uses of the confocal laser scanning microscope
The confocal laser scanning microscope is a very popular instrument in a wide variety of fields involving biological science and medical research. These include microbiology, cell biology, developmental biology and genetics. The confocal laser scanning microscope is also extremely flexible and relatively easy to use for such a sophisticated instrument, making it popular in many laboratories and research institutions. In fact, it has become the preferred instrument over the electron microscope, which was the hands-down favorite of a few years back.
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