Difference Between Fluorescence And Confocal Microscopy Pdf

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A fluorescence microscope is much the same as a conventional light microscope with added features to enhance its capabilities. Fluorescent microscopy is often used to image specific features of small specimens such as microbes. It is also used to visually enhance 3-D features at small scales.

Fluorescence Microscopy

Among the most popular tools to conduct optical sectioning is the spot-scanning laser confocal microscope where a single spot of focused light is rapidly transitioned along the lateral axes of the specimen to excite fluorescence from labeled components. Fluorescence emission gathered from the specimen is filtered through a confocal pinhole aperture to reject light that originates from regions removed from the focal plane. The emission light that passes through the pinhole is detected by a photomultiplier to form the image in a serial manner.

Although the spot-scanning approach is much slower than parallel scanning techniques such as a spinning disk , it offers great flexibility in terms of image size and acquisition strategies. In addition, laser scanning confocal microscopy is capable of producing the highest out-of-focus discrimination of all routine optical sectioning techniques. This interactive tutorial explores optical sectioning with confocal microscopy and compares these sections to the results obtained with widefield fluorescence.

In laser scanning confocal microscopy LSCM , it is possible to exclusively image a thin optical slice out of a thick specimen ranging in physical section thickness up to micrometers , a technique known as optical sectioning. Under suitable conditions, the thickness z-dimension of such a optical slice can be less than nanometers.

The fundamental advantage of the confocal versus a traditional widefield microscope arises due to the restricted manner in which light reaches the photomultiplier through a pinhole. In widefield fluorescence, the image of a thick biological specimen will only be in focus if its axial dimension is less than the wave-optical depth of focus specified by the objective parameters.

In cases where this condition is satisfied, the in-focus image information from the specimen plane of interest is mixed with out-of-focus image information arising from regions outside the focal plane. This reduces image contrast and increases the share of stray light detected.

If multiple fluorophores are being observed, there will also be a color mix of the image information obtained from all of the channels involved. Alex B. Coker and Michael W. Select a Country or Region.

Interactive Tutorials Optical Sectioning. Confocal versus Widefield Microscopy. Contributing Authors Alex B.

Confocal versus Widefield Microscopy

Among the most popular tools to conduct optical sectioning is the spot-scanning laser confocal microscope where a single spot of focused light is rapidly transitioned along the lateral axes of the specimen to excite fluorescence from labeled components. Fluorescence emission gathered from the specimen is filtered through a confocal pinhole aperture to reject light that originates from regions removed from the focal plane. The emission light that passes through the pinhole is detected by a photomultiplier to form the image in a serial manner. Although the spot-scanning approach is much slower than parallel scanning techniques such as a spinning disk , it offers great flexibility in terms of image size and acquisition strategies. In addition, laser scanning confocal microscopy is capable of producing the highest out-of-focus discrimination of all routine optical sectioning techniques. This interactive tutorial explores optical sectioning with confocal microscopy and compares these sections to the results obtained with widefield fluorescence. In laser scanning confocal microscopy LSCM , it is possible to exclusively image a thin optical slice out of a thick specimen ranging in physical section thickness up to micrometers , a technique known as optical sectioning.

Confocal microscopy offers several distinct advantages over traditional widefield fluorescence microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane that leads to image degradation , and the capability to collect serial optical sections from thick specimens. 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 dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope. The tutorial initializes with a randomly-selected specimen from Set 1 appearing in the widefield Widefield Image and confocal Confocal Image specimen image windows. When the Focus Lock checkbox is enabled, both the widefield and confocal focus sliders are simultaneously engaged to present a comparison of the images gathered at similar focal planes by each technique. The Scan Line Speed slider controls the scanning speed of the confocal image, while the Pinhole Aperture Size radio buttons can be utilized to toggle between small 1 Airy unit , medium 4 Airy units , and large 20 Airy units pinhole sizes.

Fluorescence Microscopy

Confocal Microscopy: Methods and Protocols, Second Edition takes the researcher from the bench top through the imaging process, to the page. Protocols for the preparation of tissues from many model organisms including worms, flies and mice have been included as well as chapters on confocal imaging of living cells, three dimensional analysis, and the measurement and presentation of confocal images for publication. Emphasis has been placed on the laser scanning confocal microscope since this is still the instrument used for most routine applications.

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation.

Laser scanning confocal microscopy , more generally referred as confocal microscopy, is an established microscopy technique that allows obtaining 2D or 3D high-resolution images of relatively thick samples.

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3 Response
  1. Talon Q.

    Confocal microscopy , most frequently confocal laser scanning microscopy CLSM or laser confocal scanning microscopy LCSM , is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.

  2. Prunagblacfis

    This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope.

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