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Practical:2 L ight Microscopy. presented by: Ms Asmaa A Basonbul. Objectives. Recognize different parts of a compound light microscope. Learn how using appropriate objective lenses e.g 10x for focusing 40x for wet preparation 100x oil immersion
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Practical:2Light Microscopy presented by: Ms Asmaa A Basonbul
Objectives • Recognize different parts of a compound light microscope. • Learn how using appropriate objective lenses e.g 10x for focusing 40x for wet preparation 100x oil immersion • Learn how using condenser and iris diaphragm to adjust the light source to the optimum.
Introduction Microscopy: The use of microscopes in all their various forms. Q. Why we use microscope in haematology lab? To study various blood cells and inclusion bodies.
How? By using a system of lenses and illumination sources it’s make object visible. Q. How many times the microscope can magnify that object? Microscope can magnify an object from 100-1000 times of it’s original size.
Principle To magnify the object the LM use a system of lenses (objectives and oculars) to manage the path of light beam that travel b/w object which we’re studied to the eye.
Parts of light microscope • Eye picec (ocular lenses): Magnify lens with magnification power 10x.
Cont: Parts of light microscope • Body tube: Contains mirrors and prisms that transmit the image from the objective lens to the ocular lens.
Cont: Parts of light microscope • Objective lenses: Primary lenses that magnify specimen • Low power 10x • High power 40x • Oil immersion 100x
Cont: Parts of light microscope • Stage: Holds slide in position.
Cont: Parts of light microscope • Condenser: Lens system that condenses light before it passes through the specimen.
Cont: Parts of light microscope • Iris diaphragm: Control the amount of light entering the condenser.
Cont: Parts of light microscope • Coarse and fine adjustment knobs: Used for focusing the specimen.
Cont: Parts of light microscope • Light: Source of illumination, bulb.
Types of Light Microscopy • Bright field microscopy. • Dark field microscopy. • Fluorescence microscopy. • Phase contrast microscopy.
Bright filed microscope Light bulb Day light transfer bent Object in the specimen
Dark filed microscope Special objective U Special condenser in mico Light source transfer bent Object in the specimen “Microorganism”
Specimen labeled with fluorescence dye • Acridine orange • Auramine/rhodamine
Specimen labeled with fluorescence dye • Calcofluor white
Phase contrast microscope contrast Bright(denser) Dark(lighter) special objectives Light source Picture appear with different darkness and brightness bent transfer Object in the specimen Special condenser
Materials • Compound microscope. • Lens cleaning paper/ cloth. • Immersion oil. • Wet preparation. • Stained preparation.
Oil immersion objective(100x): • Put drop of oil immersion on the slide. • Turn the oil immersion objective 1oox and put above the slide. • The 100x objective will immersed in the oil. • The picture is transmitted from objective lens to ocular lens.
The oil helps to keep light rays together as they pass b/w the specimen and objective lens.
Total magnification power of a microscope: = power of the objective lens X power magnification of eye piece e.g : using oil immersion objective 100x ocular lens (eye piece) 10x ??!! So 100x X 10x =1000
Resolving power of microscope: Is measure of its ability to discriminate b/w two adjacent objects. Absolute limit of the resolving power = wave length of LM = 400-800nm
Maintenance of microscope • The microscope is delicate instrument which is must be: • Handle gently. • Put in a clean environment away from chemical, direct sunlight, evaporation, heating source or moisture. • Cleaned immediately if the stage is contaminated with saline to avoid corrosion. • The temperate climate humidity and high temperature causes growth of fungus which can damage optical surfaces
Cleaning the microscope • Optics: • The low and high power lens paper tissue. • Oil immersion lens paper tissue and few drop from xyelen (if needed). • Non-optics: • Eyepices: Using the soft camel-hair brush to remove the dust from in and out side.
Cleaning the microscope • Condenser and iris diaphragm: Using soft cloth or tissue moistened with toluene and the mirror with 5% alcohole. Other parts cleaned with mild detergent and remove grease or oil with petroleum ether followed by 45% ethanol in water.
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