Monica Lorraine L.

Segismundo 2AMT A MICROSCOPE is an optical instrument that uses lenses to produce magnified images of small objects, especially of objects too small to be seen by the unaided eye. They are two types of microscopes; the Light Microscope, which uses visible light, and the Electron Microscope, which use electron beams. The Light Microscope employs visible light, to detect small objects. It is a well-known and well-used research tool in biology. In Histology, it is used to reveal and study tissue features. The Bright-Field, Fluorescence, Phase-Contrast, Differential Interference, Confocal, and Polarizing microscopes are examples of light microscopes. The Bright-Field microscope is the most used in Histology classes. The slides are stained and examined by means of ordinary light that passes through the specimen. This kind of light microscope is composed of both mechanical and optical parts.

Bright-Field microscope

The optical parts consist of three systems of lenses, namely the Condenser, the Objectives, and the Eye Piece. The Condenser collects and focuses light, producing a cone of light that illuminates the object to be observed. The Objective lenses enlarge and project the illuminated image of the object in the direction of the eyepiece. For studying Histology, three different magnifications are generally used, namely the X4, X10, and X40. X4 is used for low magnification observations of a large area of a tissue. X10 is used for a medium magnification of a smaller field. X40 is used for high magnification observations of more detailed areas. The Eye Piece, also called the Ocular Lens, further magnifies the image by X10 and projects the image so the viewer may be able to see this.

Pathway of light in Bright-Field Microscope

Fluorescence is when certain substances are irradiated by light on a proper wavelength; thus, they emit light with a longer wavelength. Fluorescence microscopes are used when tissue sections are irradiated with ultraviolet (UV) light, and the emission is in the visible portion of the spectrum. This type of light microscope has a strong UV light source and special filters that select rays of different wavelengths emitted by the substances. The fluorescent substances appear brilliant on a dark background.

Fluorescence microscope

Pathway of light in Fluorescence microscope

Example of specimen seen through the Fluorescence microscope

Unstained biological specimen are usually transparent and difficult to view in detail, because all parts of the specimen have almost the same optical density. Phase-contrast microscopy was first described in 1934 by Dutch physicist Frits Zernike. According to Zernike, it is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue

slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). Phase-contrast microscopes use a lens system that produces visible images from transparent objects. This is based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices. These changes are used to cause the structures to appear lighter or darker in relation to one another. Because it does not require staining or fixation of the specimen, it allows the observation of living cells and tissue cultures, which may be useful in Histology classes.

Similar to the Phase-Contrast microscope, the Differential Interference microscope renders contrast in transparent specimen. It is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount. It produces an image with a more apparent three-dimensional aspect than Phase-Contrast microscopes. Differential Interference microscopy is a technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.

Confocal microscope offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. Confocal microscopes avoids stray light and achieves greater resolution by using a small point of high-intensity light provided by a laser, and a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of the lens, and the detector's pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confocal) and unfocused light does not pass through the pinhole. This greatly improves resolution of the object in focus and allows the localization of specimen components with much greater precision than with the bright-field microscope. Most confocal microscopes include a computer-driven mirror system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly. Digital images captured at many individual spots in a very thin plane-of-focus are used to produce an "optical section" of that plane. Moreover, creating optical sections at a series of focal planes through the specimen allows them to be digitally reconstructed into a three-dimensional image.

Confocal microscope

Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials. Polarizing microscopes have a high degree of sensitivity, and can be utilized for both quantitative and qualitative studies targeted at a wide range of anisotropic specimens. This allows the recognition of structures made of highly organized molecules, such as cellulose, collagen, microtubules and microfilaments. Polarizing microscopes produces an image only of material having repetitive, periodic macromolecular structure. In polarizing microscopy, when normal light passes through a polarizing filter, it exits vibrating in only one direction. If a second filter is placed above the first one, with its main axis perpendicular to the first filter, no light passes through. If, however, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repetitive structure rotates the axis of the light emerging from the polarizer, thus, appearing as bright structures against a dark background

An Electron Microscope is an instrument that uses electronic or other processes to magnify objects. a microscope with high magnification and resolution, employing electron beams in place of light and using electron lenses. The wavelength in the electron beam is much shorter than of light, allowing a thousand-fold increase in resolution. Examples of this kind of microscope are the Transmission Electron Microscope and the Scanning Electron Microscope. The Transmission Electron Microscope is a device that transmits electrons instead of light through a specimen. The device imaging system that allows for a much higher resolution than can be obtained with a light microscope, allowing for the visualization of even a single column of atoms. This high resolution allows magnifications of up to 400,000 times to be viewed with details. The TEM is used in a range of scientific fields for applications in cancer research, virology, and materials science. To provide a useful interaction between the specimen and the electrons, TEM requires very thin sections (4090 nm). The TEM has a beam of electrons that can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. The beam is produced by a cathode at the top of the instrument and passes down through the chamber in a vacuum. Because electrons change their path when submitted to electromagnetic fields, the beam can be focused by passing through electric coils which can be considered electromagnetic lenses. The configuration of the TEM is similar to that of an upside-down light microscope. The first lens is a condenser that focuses the beam of electrons on the section. Some electrons interact with atoms of the section and continue their course, while others simply cross the specimen without interacting. Most electrons reach the objective lens, which forms a magnified image that is then projected through other magnifying lenses. Because the human eye is not sensitive to electrons, the image is finally projected on a fluorescent screen or is registered by photographic plates or a CCD camera. In a TEM image, areas of the specimen through which electrons passed appear bright (electron lucent), while those areas which are naturally dense or which bind heavy metals during specimen preparation or "staining" absorb or deflect electrons and appear dark (electron dense). Such images are therefore always black, white, and shades of gray.

Transmission Electron Microscope

Scanning Electron Microscope (SEM) permits pseudothree-dimensional views of the surfaces of cells, tissues, and organs. Like the TEM, this microscope produces and focuses a very narrow beam of electrons, but however, here the electron beam focused by electromagnetic lenses does not pass through the specimen, but rather is moved sequentially (scanned) from point to point across its surface similar to the way an electron beam is scanned across a television tube. The surface of the specimen is first dried and coated with a very thin layer of metal atoms through which electrons do not pass readily. When the beam is scanned from point to point across the specimen, it interacts with the metal atoms and produces reflected electrons or secondary electrons emitted from the metal. These are captured by a detector and the resulting signal is processed to produce a black-and-white image on a monitor. The SEM shows only surface views of the coated specimen but with a striking three-dimensional quality. The inside of organs or cells can be analyzed by sectioning them to expose their internal surfaces. SEM images are usually easily understood, because they present a view that appears to be illuminated from above, just as our ordinary macroscopic world is filled with highlights and shadows caused by illumination from above.

Scanning Electron Microscope

References: Junqueiras Basic Histology by Anthony L. Mescher http://www.microscopyu.com/ http://www.news-medical.net/ Google images