1) Light Microscope
Conventional bright-field microscopy, as well as fluorescence, phase-contrast, differential interference, confocal, and polarizing microscopy are all based on the interaction of light and tissue components and can be used to reveal and study tissue features.
Type of light Microscope
Bright-field microscopy:-
With the bright-field microscope, widely used by students of histology, stained preparations are examined by means of ordinary light that passes through the specimen. The microscope is composed of mechanical and optical parts.
The optical components consist of three systems of lenses. 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. The eyepiece or ocular lens further magnifies this image and projects it onto the viewer's retina, photographic film, or (to obtain a digital image) a detector such as a charge-coupled device (CCD) camera. The total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses.
Fluorescence microscopy:-
When certain substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength. This phenomenon is called fluorescence. In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant on a dark background. For this method, the microscope has a strong UV light source and special filters that select rays of different wavelengths emitted by the substances.
Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains. Acridine orange, which binds both DNA and RNA, is an example. When observed in the fluorescence microscope, these nucleic acids emit slightly different fluorescence, allowing them to be localized separately in cells. Other compounds such as Hoechst stain and DAPI specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV. Another important application of fluorescence microscopy is achieved by coupling fluorescent compounds to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures under the microscope. Antibodies labeled with fluorescent compounds are extremely important in immunohistological staining.
Phase-contrast Microscopy & Differential interference microscopy:-
Some optical arrangements allow the observation of unstained cells and tissue sections. Unstained biological specimens are usually transparent and difficult to view in detail, because all parts of the specimen have almost the same optical density. Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects.
Confocal Microscopy:-
With a regular bright-field microscope the beam of light is relatively large and fills the specimen. Stray light reduces contrast within the image and compromises the resolving power of the objective lens. Confocal microscopy 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.
Polarizing Microscopy:-
Polarizing microscopy allows the recognition of structures made of highly organized molecules. When normal light passes through a polarizing filter (such as a Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope 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 and they appear as bright structures against a dark background. The ability to rotate the direction of vibration ofpolarized light is called birefringence and is a feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and microfilaments.
2) Electron Microscopy
Transmission Electron Microscopy:-
The transmission electron microscope (TEM) is an imaging system that permits resolution around 3 mm. This high resolution allows magnifications of up to 400,000 times to be viewed with details. Unfortunately, this level of magnification applies only to isolated molecules or particles. very thin tissue sections can be observed with details at magnifications of up to about 120,000 times.
