Confocal microscopy

= Confocal Microscopy=

Overview
Microscopy is the use of microscopes to view and enlarge details from objects that are typically too small to be examined properly with the naked eye. There are 3 main types of microscopy: Optical, Electron and Scanning Probe microscopy. Optical microscopy is a technique that uses visible light and lenses to enlarge and create an image that can be detected by the human eye or imaged by a camera or a detector such as a photographic plate.

Fluorescent microscopy is a sub-type of optical microscopy that uses fluorescent properties of an object (or of dyes that stain the object) to obtain a highly detailed, specific image of the desired structure or molecule with colours distinct to that of the light source.

Confocal Microscopy is an optical imaging technique developed in 1957 by Marvin Minsky. It uses fluorescence to increase resolution and contrast of an image. It involves the emission of light from a laser source through a pinhole that is located on a confocal plane with a detector, where the light also passes through a pinhole aperture. The focal point at the laser’s pinhole aperture is the same as the focal point of the detector’s aperture; the device’s name comes from the principle of the two conjugate planes. The light from the laser passes through a lens, which focuses the rays onto a specific point on the specimen, called the “scanning point”. This is then reflected using a dichromatic mirror to a detector, usually a photomultiplier tube. As the light from the laser is simultaneously reflected from the dichromatic mirror and scanned by the photodetector, fluorescence from the specimen is also reflected onto the detector.

A confocal microscope offers an enhanced, fully focused image of a small point on the desired object as opposed to a wide field fluorescence microscope, where the entire object is flooded with light and a large area of the image is unfocused. This is achieved by filters that remove glare and out of focus light. Confocal microscopy allows the user to control the depth of the field and to also eliminate any background information, which can reduce the quality of image. An important feature of confocal microscopy is that has the ability to produce 3D images from optical sections of thicker specimens, which is a reason as to why it has applications in a wide range of sciences, such as clinical medicine.

Relevance to Clinical Medicine
Confocal microscopy has many clinical applications, as it has the ability to image both fixed and living cells. An important clinical application of the confocal microscope is the imaging of the cornea and conjunctiva to study the corneal structure of the eye. Confocal microscopy is a non-invasive technique, thus is a perfect fit for studying a patient’s eye. It can detect a range of pathological conditions, such as infective keratitis and corneal dystrophy. However, the technique’s major limitation when studying the eye is the back scattering of light, which has lead to several specialised confocal microscopes to be developed.

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Types of Confocal Microscopes
There are 4 Main types of confocal microscopy:

- Confocal laser scanning microscopes - Spinning-disc confocal microscopy - Micro-lens enhanced/dual spinning disc confocal microscopy - Programmable array microscopes
 * These typically use 2-3 mirrors to ‘scan’ the sample
 * These use moving pinholes on a spinning disc to illuminate specific points on the sample. As these devices use less energy than other types of confocal microscopes that use lasers, the quality of image is better for imaging live tissues.
 * Very similar to spinning disc microscopes, but contain 2 spinning discs and micro-lenses behind the pinholes. This allows more light to be focused onto each focal point on the specimen.
 * Uses an electronic device called a ‘spatial light modulator’, which contains moving pinholes and can be adjusted from a computer. A ‘Charged Couple Device’ camera then detects the resulting image.

Advantages and Limitations of Confocal Microscopy
Operating a confocal microscope is relatively easy, and images can be taken and developed quickly. The technique produces high quality images of a specific area of a specimen. A limitation of confocal microscopy is that it can lose intensity due to light passing through small pinholes; to obtain a high quality image, long exposure is needed.

Despite only being invented in 1957, confocal microscopy has become a hugely important technique across the life sciences and has been further developed into highly specialised techniques in the last 20-30 years [see above]. However, until recently many of these types (such as the spinning disc microscope) were not widely available for commercial use. As technologies advance and the demand for non-invasive, microscopic imaging of live cells in clinical practice increases, so has the production of confocal microscopes and its use around the world.