Confocal Microscopy:
We have a confocal microscope in our lab, and it is hella tight. I have been using it to analyze data concerning the fluorescence of a molecule called lipofuscin. This molecule is special because, in its final form, it cannot be broken down any further within the cell. Therefore, it starts to build up. As it builds up, it can be detected with fluorescence microscopy as "granules" within the cell. These granules are what I've been analyzing. (I still can't tell you why. Stay tuned for that.)
Some Info - (for more, go to wikipedia. That's where this came from) -
"The principle of confocal imaging was patented by Marvin Minsky in 1957.[2] In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. As only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction."
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CLSM is a scanning imaging technique in which the resolution obtained is best explained by comparing it with another scanning technique like that of the scanning electron microscope (SEM). Do not confuse CLSM with phonograph-like imaging—AFM or STM, for example, where the image is obtained by scanning with an atomic tip over a surface.
In CLSM a fluorescent specimen is illuminated by a point laser source, and each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning volume is determined by the spot size (close to diffraction limit) of the optical system because the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the numerical aperture of the system's objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using wide-field illumination. However, with confocal microscopy it is even possible to improve on the resolution limit of wide-field illumination techniques because the confocal aperture can be closed down to eliminate higher orders of the diffraction pattern. For example, if the pinhole diameter is set to 1 Airy unit then only the first order of the diffraction pattern makes it through the aperture to the detector while the higher orders are blocked, thus improving resolution at the cost of a slight decrease in brightness. In practice, the resolution limit of confocal microscopy is often limited by the signal to noise ratio caused by the small number of photons typically available in fluorescence microscopy. One can compensate for this effect by using more sensitive photodetectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination later risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required."
In conclusion, it's all about the pinhole baby.
And that is this week's science blag. Stay tuned next week where we wil discuss the most heated question in science: Was T. rex primarily a hunter or a scavenger?
Peace.
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