University:
American Career College
Course:
MICR-112 | General Microbiology
Academic year:

2020
Views:

45

Pages:

18
Author:

pitoGx

PARAMETERS USED IN MICROSCOPY 1.MAGNIFICATION .  MAGNIFICATION : - Its the enlargement of the image of the specimen. = size of an object seen under microscope actual size observed with unaided eye TOTAL MAGNIFICATION = magnification of the eye piece x magnification of the objective  Depends upon- Optical tube length -Focal length of objective -Magnifying power of the eye piece Objective lens Ocular lens Magnification Color code 4x 10x 40x Red 10x 10x 100x Yellow 40x 10x 400x Light blue 100x (oil) 10x 1000x White 2.RESOLUTION AND RESOLVING POWER  RESOLUTION : Ability of a lens to produce separate images of closely placed objects so that they can be distinguished as 2 separate entities. Shorter wavelength = greater resolution High refractive index = high resolution  RESOLVING POWER (LIMIT OF RESOLUTION – LR) : is the minimum distance between two objects which when magnified still allows the two objects to be distinguished from each other. It’s its capacity to distinguish two neighboring points as separate entities.  FIELD OF VIEW: the visible area when we look through the microscope. The minimum resolvable distance between two luminous points (r) is given by formula: where ; r =0.61×λ NA λ = wave length of the light NA = Numerical aperture TYPES OF MICROSCOPE RESOLVING POWER Human eye 0.25 millimetre Light microscope 0.25 micrometre Compound Microscope 200 nanometres Scanning Electron Microscope 10 nanometres Transmission Electron Microscope 0.2 nanometres 3.NUMERICAL APERTURE  Capacity of an objective to render the outline of the image of an object clear and distinct.  It’s the ratio of diameter of lens to its focal length: NA = n Sin θ/2 n = refractive index, θ = angle of aperture (CAD)  n of air = 1 , n of water=1.33, n of oil = 1.52  Depends upon elimination of chromatic and spherical aberration.  The numerical aperture is greater for higher magnification objectives. • 10x=0.25 mm • 40x=0.65 mm • 100x=1.30mm 4.WORKING DISTANCE: Distance from the front lens element of the objective to the closest surface of the coverslip. • 10x= 5-6mm • 40x= 0.5-1.5mm • 100x= 0.15-0.20mm 5.CONTRAST • Difference in the intensity between two objects or between an object and the background. 6. REFRACTIVE INDEX: Determines direction and magnitude of bending. It is ratio of speed of light in one medium to another. 7. FOCAL POINT: Light rays focus at a specific point in the lens. 8.FOCAL LENGTH: Distance between center of lens and focal point. Strength of lens is related to focal length. Short focal length ⇒ more magnification 9.ABBERATION Failure of rays to converge at one focus because of defect in lens or mirror. Optical Aberration Spherical aberration Chromatic aberration A. SPHERICAL ABBERATION  Its is due to the fact that rays passing through the edge of the lens will seldom be brought to precisely the same focus as those passing nearer the centre, the result may be serious distortion of image. B. CHROMATIC ABBERATION When the white light passes through a lens its diffracted as in a prism to its component colors, each with its own wavelength. • The rays of different wavelengths are refracted by the lens to varying extent and may not always be recombined in the same focus. • The result may be a hazy image fringed with the colours of the spectrum. • PARTS OF MICROSCOPE • It consists of mainly three parts: Mechanical part - base, C-shaped arm and stage. Magnifying part - objective lens and ocular lens.  Illuminating part - sub stage, condenser, iris diaphragm, light source. Adjustment knob: 1. Fine adjustment knob 2. Coarse adjustment knob MECHANICAL PARTS: MECHANICAL STAGE: To place the slide.  STAGE CLIP: Hold the slide.  STAGE APERTURE: For passing the light.  TUBE: Connect eyepiece and objective lens. 160 mm in length.  ARM: Connect tube to the base and used for holding.  BASE: For support . MAGNIFYING PARTS:A.OBJECTIVE LENS:  It forms magnified real image.  Mounted on Nose piece.  10X (Low power field, total magnification of 100X,for examination of protozoa and larger organism)  40X (High power lens, total magnification 400X,for examination of bacterial motility, protozoa ,fungi)  100X (Oil immersion, largest numerical aperture, smallest lens size, total magnification 1000X, resolution 0.2 micron ) 4 TYPES OF OBJECTIVE LENSES: 1.ACHROMATIC- corrected for: 2-colour chromatic aberration & 1-colour spherical aberration. -Used for routine work and does not require high resolution. 2.FLUORITE(FL)- well corrected for chromatic & spherical aberration. -not corrected for field curvature. -Suitable for Photomicrography & high power work. 3.PLAN-ACHROMATIC(PLAN)- corrected for field curvature. - Suitable for Photomicrography. 4.PLAN-APOCHROMATIC(PLAN-APO)- corrected for field curvature. - 3-colour chromatic, 2-colour spherical aberration. - Provides excellent whole flat field images and high resolution. - Ideal for colour Photomicrography. - Their high cost may not be justified in routine laboratory setting. OIL IMMERSION OBJECTIVE: 1. Highest magnification 2. Oil prevents refraction of light outwards and allows it to pass straight in to objective. B.EYE PIECE  Forms magnified, virtual & erect image  TYPES (a) Monocular (b) Binocular (c) Trinocular  ILLUMINATING OR (a) Huygenian (b) Ramsden (c) Compensating PARTS:- MIRROR : Plano-concave , reflects light from external source. DIAPHRAGM/IRIS : Controls intensity of light. CONDENSER : Focuses light from the illuminate source on object. ILLUMINATION SYSTEM OF MICROSCOPE  A quartz halogen illumination is preferred to a tungsten illumination because it gives a consistent bright white illumination.  A tungsten illumination gives a yellow light and its intensity becomes less as the bulb blackens.  Low wattage lamps are used eg. 6V 20W or 6V 10W  Recently LED bulbs have come to use. They have overcome the limitations of above mentioned sources .  Their advantages are-they last for a very long time -extremely low in heat emission Adjustment knob: COARSE ADJUSTMENT  FINE ADJUSTMENT INTERPUPILLARY AREA REVOLVING NOSE PIECE/TURRET: 3 or more objective lenses are attached & rotated for changing the power of objective lens.