Confocal Microscopy

Confocal Microscopy

Confocal microscopy offers several advantages over conventional wide field optical microscopy. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional fluorescence microscopy, and the growing number of applications in cell biology that rely on imaging both fixed and living cells and tissues. In fact, confocal technology is proving to be one of the most important advances ever achieved in optical microscopy.

Principles of confocal microscopy
• a focused laser beam serves as a high intensity point source
• light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane
• a sensitive detector (photomultipler) behind a pinhole to measure the intensity of light
• the laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor

Application in biomedical science
• Growth of small organisms, cells, embryos
• Movement of intracellular structures
• Change in membrane permeability
• 3 dimensional reconstruction
• Image analysis


? Fluorescence Microscopes
A specialized type of optical microscope, called afluorescence microscope, uses fluorescence(ultraviolet light as its light source) to create an image. In a fluorescence microscope, specimens are first stained with a fluorescent dye that binds specifically to certain compounds in the specimen. Then, light of a single wavelength is used to cause the fluorescent dyes to give off light that can be picked up by a detector.
Fluorescent microscopes are very useful in the life sciences where they are used to visualize the expression of specific proteins or other molecules inside cells.
Also a laboratory technique called the fluorescent?antibody technique employs fluorescent dyes and antibodies to help identify unknown bacteria.

In this endothelial cell, fluorescence microscopy shows nuclei
(stained blue), microtubules (stained green), and actin filaments (stained red).

? Electron Microscope
What is Electron Microscopy?
The electron microscope is a type of microscope that uses a beam of electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail. They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring trained personnel to operate them.
? Types of Electron Microscopes :
• Transmission Electron Microscope (TEM)
In a transmission electron microscope or TEM, a beam of electrons hits a very thin sample (usually no more than 100 nm thick). The electrons are transmitted through the sample (Figure 2). After the sample, the electrons hit a fluorescence screen that forms an image with the electrons that were transmitted. You can better understand this process by imagining how a movie projector works. In a projector, you have a film that has the negative image that will be projected. The projector shines white light on the negative and the light transmitted forms the image contained in the negative.
• Scanning Electron Microscope (SEM)
In a scanning electron microscope or SEM, a beam of electrons scans the surface of a sample . The electrons interact with the material in a way that triggers the emission of secondary electrons. These secondary electrons are captured by a detector, which forms an image of the surface of the sample. The direction of the emission of the secondary electrons depends on the orientation of the features of the surface. There, the image formed will reflect the characteristic feature of the region of the surface that was exposed to the electron beam.


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