Defining the MicroscopeA microscope, whether simple or compound, is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye. The simplest microscope is a magnifying glass or a pair of reading glasses.

Bright-Field MicroscopeThe compound bright-field microscope is the direct descendant of the microscopes that became widely available in the 1800s and opened the first major era of histologic research. It has become the most common tool of histology and histopathology. The term compound refers to a series of lenses while bright-field means that the entire field is illuminated by an ordinary condenser. Specimens must be translucent and stained to provide contrast.

Essential Components of a Microscope light source for illumination of the specimen condenser lens to collect light from the source and project it as a cone through the specimen stage on which the slide or other specimen is placed objective lens to enlarge and resolve the specimens image and project it toward the ocular lens ocular lens to further enlarge the image and project it onto the observers retina

Optical Properties of Lenses Magnification increases the specimens apparent size and makes it appear closer. The total magnification value is obtained by multiplying the power of the objective lens by that of the ocular lens. Numerical aperture (NA) is related to the width of the lens opening. The greater the NA is, the greater is the resolving power. Refractive index measures the comparative velocity of light in different media. The air between the lens and the coverslip bends some of the light projected through the specimen. At high magnifications, the accompanying loss of resolution reduces image quality. To maintain the refractive index and thus improving the resolution, use an immersion oil between the coverslip together with an oil immersion objective lens. Resolution measures how close two objects can be and still appear separate. The smaller the value, the greater the resolution. Increased magnification is useless without improved resolution. Resolution is independent of magnification and is calculated from the numerical aperture (NA) of the objective and the wavelength () of illumination:

The resolving power of the human eye (0.2 mm) is determined by the spacing of the photoreceptor cells in the retina. The role of a microscope is to magnify an image to a level at which the retina can resolve the information that would otherwise be below its limit of resolution.

Other Types of MicroscopesBesides bright-field microscopy, which is commonly used for routine examination of histologic slides, other optical systems are used in clinical and research laboratories. They usually vary based on wavelength of specimen illumination, physical alteration of the light coming to or leaving the specimen, and specific analytic processes that can be applied to the final image.

1. Dark-Field Microscope

In dark-field microscopy, no direct light from the light source is gathered by the objective lens.This microscope uses a special condenser to provide contrast in unstained material. A disk-like shield excludes the center of the light shaft from the condenser so that the specimen is illuminated from the sides. Objects that deflect light into the objective lens are visible and appear bright on a dark background.The dark-field microscope is useful in examining autoradiographs, in which the developed silver grains appear white in a dark background. Clinically, dark-field microscopy is useful in examining urine for crystals, such as those of uric acid and oxalate, and in demonstrating specific bacteria such as spirochetes, particularly Treponema pallidum, the microorganism that causes syphilis, a sexually transmitted disease.

2. Phase-Contrast Microscope

The phase contrast microscope enables living specimens to be visualized and allows examination of unstained cells and tissues.It uses a special lens system to transform invisible differences in phase retardation into visible differences in light intensity. Where the lights are "in-phase" the image is brighter, where the lights are "out of phase" the image is darker, and by amplifying these differences in the light, it enhances contrast. Fixation and staining are unnecessary. However, specimens must be thin and translucent. High resolution is difficult to obtain. In addition, phase-contrast microscope is used extensively to examine unstained semithin (approximately 0.5-m) sections of plastic-embedded tissue.

Two Modifications of the Phase Contrast Microscopea. Interference Microscope

This type allows quantification of tissue mass by applying the principle that refractive index and phase retardation are proportionate to mass.This combines the optical features of phase contrast and polarizing microscopes to provide contrast in unstained material. It can measure the phase retardation induced by components of a specimen by relying on differences in refractive index. Unlike standard phase contrast microscopes, an interference microscope can compare the refracted light with an unimpeded reference beam and provide an electronic readout of the data.b. Differential Interference Microscope

It uses Nomarski optics which is especially useful for assessing surface properties of cells and other biologic objects.3. Polarizing Microscope

The polarizing microscope uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light.The polarizing microscope is a simple modification of the light microscope in which a polarizing filter is located between the light source and the specimen, and a second polarizer (the analyzer) is located between the objective lens and the viewer.Both the polarizer and the analyzer can be rotated; the difference between their angles of rotation is used to determine the degree by which a structure affects the beam of polarized light.The ability of a crystal or paracrystalline array to rotate the plane of polarized light is called birefringence (double refraction). Striated muscle and the crystalloid inclusions in the testicular interstitial cells exhibit birefringence. Birefringent structures appear as bright, often colored, objects on a dark background. Staining is not required.

4. 5. Fluorescence Microscope

The fluorescence microscope makes use of the ability of certain molecules to fluoresce under ultraviolet light.A molecule that fluoresces or emits light of wavelengths in the visible range when exposed to an ultraviolet (UV) source is called a fluorochrome. An excitation filter between the light source and the specimen filters out all wavelengths except that needed to stimulate the fluorochrome. A barrier filter between the objective and ocular lenses protects the eyes from UV rays and projects only the emitted light.The fluorescence microscope is used to display naturally occurring fluorescent (autofluorescent) molecules such as vitamin A and some neurotransmitters. Because autofluorescent molecules are not numerous, however, the microscopes most widespread application is the display of introduced fluorescence, as in the detection of antigens or antibodies in immunocytochemical staining procedures.

6. 7. Ultraviolet Microscope

The ultraviolet microscope uses quartz lenses with an ultraviolet light source.The image in the ultraviolet (UV) microscope depends on the absorption of UV light by molecules in the specimen. The UV source has a wavelength of approximately 200 nm. Thus, the UV microscope may achieve a resolution of 0.1 m. In principle, UV microscopy resembles the workings of a spectrophotometer; the results are usually recorded photographically. The specimen cannot be inspected directly through an ocular because the UV light is not visible and is injurious to the eye.The method is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides. It is also useful for detecting proteins that contain certain amino acids. Using specific illuminating wavelengths, UV spectrophotometric measurements are commonly made through the UV microscope to determine quantitatively the amount of DNA and RNA in individual cells.

8. 9. Confocal Scanning Microscope

The confocal scanning microscope combines components of a light optical microscope with a scanning system to dissect a specimen optically. It allows visualization of a biologic specimen in three dimensions without cutting sections.To penetrate the thicker specimen, a narrow laser beam is focused at a specific depth and scanned across the specimen. The major difference between a conventional and a confocal microscope is the addition of a detector aperture (pinhole) that is conjugate with the focal point of the lens; therefore, it is confocal. This precisely positioned pinhole allows only in-focus light to pass into a photomultiplier device, whereas the out-of-focus light is blocked from entering the detector, thus building up a sharp image of the focal plane. A series of optical sections at different depths can be stored digitally which permits reconstruction of a sharp three-dimensional image.

a. This diagram shows the path of the laser beam and emitted light when the imaging structure is directly at the focus of the lens. The screen with a pinhole at the other side of the optical system of the confocal microscope allows the light from the structure in focus to pass through the pinhole. The light is then translated into an image by computer software. Because the focal point of the objective lens of the microscope forms a sharp image at the level at which the pinhole is located, these two points are referred to as confocal points.b. This diagram shows the path of the laser beam and the emitted light, which is out of focus in relation to the pinhole. Thus, the light from the specimen that gets blocked by the pinhole is never detected.

10. Stereo Microscope

A stereo microscope is mainly used for dissection or inspection.It is the most common type next to the light microscope. Its magnification ranges from 10x to 80x. Low power is used for examining larger objects such as insect parts, plant parts, rocks, and fossils. There are two separate light paths which produce a three dimensional image of the specimen. The stereo microscope utilizes exterior light sources such as sunlight or desk lamps to illuminate the specimen. In contrast to the compound light microscope, a stereoscope produces an upright and normal image.

REFERENCESBooksGartner, L.P & Hiatt, J.L. (2009). Color atlas and text of histology. 6th ed. Philadelphia: Lippincott Williams & Wilkins.Mescher, A.L. (2013). Junqueiras basic histology. 13th ed. New York: McGraw Hill Companies, Inc.Paulsen, D.F. (2010). Histology and cell biology: Examination and board review. 5th ed. New York: McGraw Hill Companies, Inc.Ross, M.H. & Pawlina, W. (2011). Histology: A text and atlas with correlated cell and molecular biology. 6th ed. Philadelphia: Lippincott Williams & Wilkins.

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