MICROSCOPE BY Dr NEETHA

CONTENTS  HISTORY  PROPERTIES  TYPES  PARTS  WORKING PRINCIPLE

HISTORY OF MICROSCOPES

PROPERTIES OF A MICROSCOPE  Resolution- The resolving power is the distance that must separate 2 point sources of light, if they are to be seen as two distinct images.  Contrast  Magnification

TYPES OF MICROSCOPE  Bright field or Light microscope  Dark field microscope  Phase contrast microscope  Fluorescence microscope  Electron microscope

PARTS OF LIGHT MICROSCOPE Base C-shaped arm stage Mechanical Ocular lens Objective lens magnifying Condenser Iris diaphragm Light source Fine & coarse adjustment knobs Illuminating

BASE- Holds the light source, the fine and coarse adjustment knobes C- shaped arm –It holds the microscope and it connects the ocular lens to the objective lens Mechanical stage- The arm bears the stage with stage clips and stage control knobs to move the slide. It has an aperture at the center that permits light to reach the object.

Ocular lens- The arm contains an eyepiece that bears an ocular lens of 10x magnification power. Microscopes with 2 eye pieces are called binocular microscopes. Objective lens- The arm also contains a revolving nosepiece that bears 3 to 5 objectives with lenses of different magnifying power.

Condenser-It is mounted beneath the stage which focuses a cone of light on the slide. Iris diaphragm-It controls the light that passes through the condenser. Light source- It may be a mirror or an electric bulb. Fine and Coarse adjustment knobs- They sharpen the image.

DARK FIELD MICROSCOPE The object appears bright against a dark background with the help of a special dark field condenser. PRINCIPLE- The dark field condenser has a central opaque area that blocks light from entering the objective lens directly and has a peripheral annular area which allows the light to pass obliquely.

Only the light which is reflected by the specimen enters the objective lens. As a result, the specimen is brightly illuminated; but the background appears dark. APPLICATIONS It is used to identify living, unstained cells and thin bacteria like spirochetes which cannot be visualized by light microscopy.

WORKING PRINCIPLE

PHASE CONTRAST MICROSCOPE The microscope visualizes the unstained living cells by creating differences in contrast between the cells and water. It converts slight differences in refractive index and cell density into easily detectable variations in light intensity. Contrast can also be enhanced by staining the specimen.

PRINCIPLE – The condenser is similar to that of dark field microscope, consists of an opaque central area with a thin translucent ring, which produces a hollow cone of light. Light rays condenser specimen Phase ring objective lens ocular lens

APPLICATIONS OF PHASE CONTRAST Microbial mobility Determining shape of living cells Detecting bacterial components, such as endospores and inclusion bodies

FLUORESCENCE MICROSCOPE PRINCIPLE When fluorescent dyes are exposed to ultraviolet rays, they become excited and are said to fluoresce, i.e. they convert this invisible, short wavelength rays into light of longer wavelength.

APPLICATIONS OF FLUORESCENCE MICROSCOPE 1. EPIFLUORESCENCE MICROSCOPE It is the simplest format of fluorescence microscope Auto fluorescence –eg: cyclospora Microbes coated with fluorescent dye- Acridine orange –For detection of malarial parasite by QBC examination. Auramine phenol- For tubercle bacilli

IMMUNOFLUORESCENCE It uses fluorescent dye tagged immunoglobulins to detect cell surface antigens or antibodies bound to cell surface antigens. They are of 2 types 1. Direct immunofluorescence 2. Indirect immunofluorescence

ELECTRON MICROSCOPE It uses accelerated electrons as the source of illumination Wavelength of electrons can be upto 1 lakh times shorter than that of visible light photons It has better resolving power than light microscope It can reveal the details of flagella, fimbriae and intracellular structures of a cell.

TYPES OF ELECTRON MICROSCOPE o Transmission electron microscope- most common type. o Scanning electron microscope – It has been used to examine the surfaces of microorganisms in great detail. It differs from TEM, in producing an image from electrons emitted by an object’s surface rather than from transmitted electrons.

SCANNING ELECTRON MICROSCOPE

PRINCIPLE OF ELECTRON MICROSCOPE Electrons are generated by electron gun, which travel in high speed The medium of travel in EM should be a fully vaccum path. Electrons pass through a magnetic condenser and then bombarded on thin sliced specimen mounted on the copper slide.

The specimen scatters electrons passing through it. The electron beam is then focused by magnetic lenses to form an enlarged, visible image of the specimen on a fluorescent screen. Denser region in the specimen scatters more electrons and appears darker in the image since fewer electrons strike that area of the screen. Electron transparent regions are brighter.

DIFFERENCE BETWEEN LM & EM FEATURESLIGHT MICROSCOPE ELECTRON MICROSCOPE HIGHEST MAGNIFICATION About Over 100,000 BEST RESOLUTION 0.2 µm0.5 nm RADIATION SOURCE Visible lightElectron beam MEDIUM OF TRAVEL AirHigh vaccum SPECIMEN MOUNT Glass slideMetal grid ( usually copper) TYPE OF LENSGlassElectromagnet

CARE OF THE MICROSCOPE When carrying your microscope, hold it by the base and the metal support arm. When using your microscope and adjusting the focus you will need to lower the objective lens down as far as it will go. If using immersion oil, always ensure the objectives are cleaned immediately after use. Objective, eyepieces and condenser may be removed for cleaning.

When turning the microscope on and off, use the switch not the power point. Store in a clean, dry place Only use special lens paper or wipes for cleaning the lenses.