|
Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. The key feature of confocal microscopy is its ability to produce blur-free images of thick specimens at various depths. Images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece. The principle for this special kind of microscopy was developed by Marvin Minsky in 1953, but it took another thirty years and the development of lasers for confocal microscopy to become a standard technique toward the end of the 1980s. Image resolution describes the detail an image holds. ...
Confocal microscopy is an imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane. ...
Marvin Lee Minsky (born August 9, 1927), sometimes affectionately known as Old Man Minsky, is an American cognitive scientist in the field of artificial intelligence (AI), co-founder of MITs AI laboratory, and author of several texts on AI and philosophy. ...
1953 (MCMLIII) was a common year starting on Thursday. ...
Experiment with a laser (likely an argon type) (US Military) In physics, a laser is a device that emits light through a specific mechanism for which the term laser is an acronym: light amplification by stimulated emission of radiation. ...
The 1980s refers to the years from 1980 to 1989. ...
Image formation
In a laser scanning confocal microscope a laser beam passes a light source aperture and then is focused by an objective lens into a small (ideally diffraction-limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole the fluorescent light is detected by a photo-detection device (photomultiplier tube (PMT) or avalanche photodiode) transforming the light signal into an electrical one which is recorded by a computer. Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ...
A compact fluorescent lamp A fluorescent lamp is a type of electric lamp that excites argon and mercury vapor to create luminescence. ...
A beam splitter is an optical device, that splits a beam of light in two. ...
Photomultipliers, or photomultiplier tubes (PMT) are extremely sensitive detectors of light in the ultraviolet, visible and near infrared. ...
Avalanche photodiodes (APDs) are photodetectors that can be regarded as the semiconductor analog to photomultipliers. ...
Principle of a confocal microscope. The detector aperture obstructs the light that is not coming from the focal point, as shown by the dotted grey line in the image. The out-of-focus points are thus suppressed: most of their returning light is blocked by the pinhole. This results in sharper images compared to conventional fluoresence microscopy techniques and permits one to obtain images of various z axis planes (z-stacks) of the sample. Image File history File links Confocalprinciple. ...
Image File history File links Confocalprinciple. ...
The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane using one or more (servo-controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.
In addition, confocal microscopy provides a significant improvement in lateral resolution and the capacity for direct, non-invasive serial optical sectioning of intact, thick living specimens with an absolute minimum of sample preparation. As laser scanning confocal microscopy depends on fluorescence, a sample usually needs to be treated with fluorescent dyes to make things visible. However, the actual dye concentration can be very low so that the disturbance of biological systems is kept to a minimum. Some instruments are capable of tracking single fluorescent molecules. Additionally transgenic techniques can create organisms which produce their own fluorescent chimeric molecules. (such as a fusion of GFP, Green fluorescent protein with the protein of interest). GFP ribbon diagram from PDB database The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. ...
Resolution enhancement by the confocal principle Laser scanning confocal microscopy (LSCM) is a scanning imaging technique in which the resolution obtained is best explained by comparing it with another scanning technique like Scanning electron microscope (SEM). Not to be confused with phonograph-like imaging—AFM or STM, for example, where the image is obtained by scanning with an atomic tip over a conducting surface. Angular resolution describes the resolving power of any optical device such as a telescope, a microscope, a camera, or an eye. ...
SEM Cambridge S150 at Geological Institute, University Kiel, 1980 SEM opened sample chamber The scanning electron microscope (SEM) is a type of electron microscope capable of producing high resolution images of a sample surface. ...
Topographic scan of a glass surface The atomic force microscope (AFM) is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. ...
Image of substitutional Cr impurities (small bumps) in the Fe(001) surface. ...
In LSCM 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. This is due to the fact that 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 overcome this resolution limit of wide-field illuminating techniques as only light generated in a small volume element is detected at a time. Here it is very important to note that the effective volume of light generation is usually smaller than the volume of illumination; that is, the diffraction pattern of detectable light creation is sharper and smaller than the diffraction pattern of illumination. In other words, the resolution limit in confocal microscopy depends not only on the probability of illumination but also on the probability of creating enough detectable photons, so that the actual addressable volume being associated with a generated light intensity is smaller than the illuminated volume. Depending on the fluorescence properties of the used dyes, there is a more or less subtle improvement in lateral resolution compared to conventional microscopes. However, by using light creation processes with much lower probabilities of occurrence such as second harmonic generation (SHG), the volume of addressing is reduced to a small region of highest laser illumination intensity resulting in a significant improvement in lateral resolution. Unfortunately, the probability decrease in creation of detectable photons has a bad effect on the signal to noise ratio. This can be compensated by using more sensitive photo-detectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination latter risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required. The intensity pattern formed on a screen by diffraction from a square aperture Diffraction refers to various phenomena associated with wave propagation, such as the bending, spreading and interference of waves passing by an object or aperture that disrupts the wave. ...
Numerical aperture is a technical term of multiple uses: Numerical aperture of optical telecommunication fiber Numerical aperture in microscopy This is a disambiguation page — a navigational aid which lists other pages that might otherwise share the same title. ...
Second Harmonic Generation (SHG) is a subcategory of nonlinear optics in physics. ...
Uses Confocal microscopy is clinically used in the evaluation of various eye diseases. It is particularly useful for imaging, qualitative analysis and quantitafication of endothelial cells of the cornea. It is used for localising and identifying presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy. The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber, providing most of an eyes optical power [1]. Together with the lens, the cornea refracts light and, as a result, helps the eye to focus. ...
The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber, providing most of an eyes optical power [1]. Together with the lens, the cornea refracts light and, as a result, helps the eye to focus. ...
A fungal keratitis is an inflammation of the eyes cornea (called keratitis) that results from infection by a fungal organism. ...
Confocal microscopy is also used as the data retrieval mechanism in some 3D optical data storage systems. 3D Optical Data Storage is characterized by the ability to inscribe data within the volume of a data storage medium with three-dimensional resolution, as opposed to the two-dimensional resolution afforded by, for example, magnetic tape or CD. This innovation potentially allows very high data densities, but requires addressing...
See also Two-photon excitation microscopy is a technique that allows imaging living tissue up to a depth of one millimeter. ...
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed. ...
A Fluorescence Microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. ...
Stimulated Emission Depletion microscopy, or STED microscopy, is a technique that attempts to overcome the limits imposed by diffraction with standard confocal laser scanning microscopes. ...
External links |
Feeds |
Contact