Confocal+laser+scanning+microscopy+(CLSM)

__**Confocal Laser Scanning Microscopy (CLSM) **__

**Basic Description and Purpose **
Confocal Laser Scanning Microscopy (CLSM) is used to obtain high-resolution optical images at specified depths on a target specimen. This is done using controlled and precise depth of focus to select a single focal plane to focus on. The CLSM scans across a specimen point by point at the selected focal plane [4]. The process of designating focal planes and producing images at various depths is known as optical sectioning. By capturing multiple images of focal layers at various depths and imposing them in the z-axis, it is possible to reconstruct three-dimensional images of the specimen [4]. This process is done on a computer, where images are processed and then the computer creates the 3-D image. CLSM can generate high resolution topographical profiles of a specimen by creating images at one depth level at a time.

Operation
A Single wavelength source of light, induced by a laser, is passed through a source aperture and focused by an optical lens onto the sample. Molecules of the sample interact differently (absorption, emission, diffraction, scattering, fluorescence) with the incoming radiation, and secondary radiation is emitted by the sample. This secondary radiation follows the same path as the inducing radiation as it passes through the optical lens, but is filtered out by a beam splitter which redirects the radiation through an aperture to a detector. The detector is often a Photo-Multiplier Tube (PMT) that amplifies the incoming signal and converts it into an electrical signal that can be processed by a computer. The computer then generates an image of each point as the CLSM scans across the focal plane of the sample [4].


 * http://en.wikipedia.org/wiki/File:Confocalprinciple_in_English.svg **


 * Origin and History **

The original idea of this type of microscopy was patented by Marvin Minsky in 1957, but not until three decades later was it actually applied as the development of lasers was still in process. This type of microscopy is very beneficial in optical diseases, as it can break down the eye layer by layer to find infected areas. It also can be used for determining the age of Magdalen papyrus, which is a type of Egyptian paper.


 * Recent Research **

In a study investigating the sterilization of Enterococcus faecalis infected dentinal tubules, CLSM was used as a noninvasive technique to determine the efficacy of sterilization [1]. Infection of dentinal tubules was accomplished by centrifugation of the bacterial suspension into the tubules. The inoculation procedure was followed by exposure to several concentrations of NaOCl sterilization solutions. Viability staining coupled to CLSM was used to determine the proportion of living a dead bacteria in the tubules layer by layer [1].

Uranium exhibits fluorescent properties, in biological samples, cells, and biofilms, it is of significant interest to determine the role of uranium in biological systems [2]. Laser Induced Fluorescence Spectroscopy (LIFS) provides evidence of uranium fluorescence emission, but no spatial localization can be determined. CLSM was used coupled to LIFS in order to determine spatially within biological systems where uranium was occurring. This research provided evidence of extracellular and intracellular compartmentalization of uranium in biological systems.

Microfiltration of solutions through membranes result in decreased performance of membrane filtration over time due to membrane fouling [3]. Membrane fouling, the deposition of solute particles onto the membrane surface regardless of nominal pore size, is a major obstacle in the way of proliferation of this technology. CLSM coupled to fluorescence staining was used to develop an entire cross-sectional profile of microfiltration membranes as fouling occurred. a pore blockage model is currently being developed based on this research [3].