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    Microscope

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    firaoltolani
    This document provides information about microscopy and the use of light microscopes. It describes the key parts of light microscopes including the mechanical, magnifying, and illuminating parts. The document explains how magnification and resolving power work for light microscopes. It also provides guidance on using light microscopes properly and maintaining them.

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    Microscope

    Uploaded by

    firaoltolani
    10 views39 pages
    This document provides information about microscopy and the use of light microscopes. It describes the key parts of light microscopes including the mechanical, magnifying, and illuminating parts. The document explains how magnification and resolving power work for light microscopes. It also provides guidance on using light microscopes properly and maintaining them.

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    5. Microscope

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    This document provides information about microscopy and the use of light microscopes. It describes the key parts of light microscopes including the mechanical, magnifying, and illuminating parts. The document explains how magnification and resolving power work for light microscopes. It also provides guidance on using light microscopes properly and maintaining them.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    10 views39 pages

    Microscope

    Uploaded by

    firaoltolani
    This document provides information about microscopy and the use of light microscopes. It describes the key parts of light microscopes including the mechanical, magnifying, and illuminating parts. The document explains how magnification and resolving power work for light microscopes. It also provides guidance on using light microscopes properly and maintaining them.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 39

    King George Medical University, U.P.

    , Lucknow
    Faculty of Paramedical Sciences

    Topic- Microscope
    Presented By-
    Shivani Verma
    Demonstrator (DMLT)
    Faculty of Paramedical Sciences
    KGMU, Lucknow
    Father of Microscopy
    Antonie Van Leeuwenhoek (1632-1723)
    Introduction

    • Microscope is the most commonly used apparatus in the diagnostic


    laboratory.

    • It produces greatly enlarged and well-defined images of minute


    objects.
    • Micro = small
    • Scope = to view.
    • Microscope was evolved by Leeuwenhoek in 1683 in the form of hand
    ground lenses.

    • Today, a range of different microscopes from simple magnifying lenses


    to electron microscopes are utilized for various purposes in the
    medical and biological studies.

    • Most commonly used is a light microscope.


    Light Microscope

    • The usual type of microscope used in clinical laboratories is called


    light microscope that employs visible light as a source of light.

    • A light microscope can be a simple or a compound microscope.


    Simple Microscope

    • This is a simple hand magnifying lens.


    • The magnification power of hand lens is from 2x to 200x.
    Compound microscope

    • This has a battery of lenses fitted in a complex instrument. One type


    of lens remains near the object (objective lens) and another type of
    lens near the observer's eyes (eyepiece lens).
    • The eyepiece and objective lenses have different magnifications.
    • The compound microscope can be monocular having single eyepiece
    or binocular which has two eyepieces.
    Monocular & Binocular Compound Microscopes
    Parts of A Compound Microscope

    Broadly, the parts of compound microscopes are divided into three


    groups:
    1. Mechanical parts
    2. Magnifying parts
    3. Illuminating parts
    Mechanical Parts

    The mechanical parts consist of the following components:

    • Tube –
    ➢The tube can be straight and fixed or bent and movable.
    ➢The tube is hollow and holds the objectives at the lower end (near
    the object) and the eyepiece at the upper end (near the eye).

    • Body –
    ➢The body gives support to the tube.
    • Coarse Adjustment –
    ➢The coarse adjustment is controlled by a pair of large knobs one on
    each side of the body.
    ➢On rotating this knob, the tube moves with lenses.
    ➢The coarse adjustment is used with the low power (10x) objective.

    • Fine Adjustment –
    ➢The fine adjustment is used with the high power (40X) and oil
    immersion (100X) objectives.
    ➢This knob is present just below the course adjustment on either side
    of the body.
    ➢It gives clarity in the observation.
    • Stage –
    ➢The stage is a part of microscope where specimen to be observed is
    kept.
    ➢This part is under focus.
    ➢It has a pair of clips, capable of moving the object horizontally as well
    as vertically.

    • Arm –
    ➢The arm connects the body and the stage.
    ➢It is used to hold the microscope while putting from one place to
    another.
    • Substage –
    ➢The substage is a part present below the stage.
    ➢It holds the illuminating parts.

    • Foot –
    ➢On the foot, other parts of microscope are placed.
    ➢It is horseshoe shaped.
    Magnifying Parts

    The magnifying parts consist of objectives and eyepiece.

    • Objectives –
    ➢The objective stays near the object, at the bottom of the tube.
    ➢There are a group of three objectives:
    1. Low power (10X)
    2. High power (40X)
    3. Oil immersion (100X).
    ➢These objectives are attached to a revolving nosepiece that helps in
    the selection of required magnification.
    ➢The low power objective is the smallest in size, with a green ring.
    ➢The high power objective is also known as high dry objective.
    ➢It has a yellow ring.
    ➢The oil immersion objective has tiny little lens which always requires
    a drop of oil to observe the object.
    ➢It has a red ring for easy identification.
    • Eyepiece –
    ➢The eyepiece is another system of lens, which is attached to the top
    of the microscope.
    ➢The eyepiece is near to the eye.
    ➢The commonly used eyepiece has magnification 10X.
    ➢It is also available in 5X and 15X.
    ➢Some microscopes are available with only one eyepiece called
    monocular; and the other is binocular, which has two eyepieces.
    ➢The total magnification of the image of object is the product of
    magnifying power of eyepiece and that of objective.
    Illuminating Parts

    • The illumination system provides proper illumination to the object.


    • It involves the following components.

    • Light Source –
    ➢Most modern microscopes have a built-in internal light source with
    an electric lamp.
    ➢It provides better control of illumination.
    ➢The external light source can be an external lamp or from the sun.
    • Mirror –
    ➢In case of the external light source, the mirror is required to reflect
    the light beam from the source to the iris and condenser.
    ➢It usually has two mirrors, mounted back to back.
    ➢One side of the mirror is concave and the other side is flat.
    ➢The concave side is used without the condenser and the flat side is
    used with the condenser.
    • Iris Diaphragm –
    ➢The iris diaphragm is located below the stage between the mirror and
    the condenser.
    ➢Its function is to regulate the amount of light.
    ➢According to the necessity, the diaphragm can be closed for less light
    or opened for more light.
    • Condenser –
    ➢The condenser is located at immediately under the stage.
    ➢It is made up of a system of convex lenses, which focus light from the
    illuminating source on the plane of the object.
    ➢The height of the condenser can be adjusted by rotating a knob,
    present on one side below the stage.
    Magnification and Resolving Power of Light
    Microscope

    • Magnification power of the microscope is the degree of image


    enlargement.
    • It depends upon the following:
    i. Length of the optical tube
    ii. Magnifying power of the objective lens used
    iii. Magnifying power of the eyepiece
    • With a fixed tube length of 160 mm in majority of standard microscopes,
    the magnification power of the microscope is obtained by the following:
    Magnifying power of objective x Magnifying power of eyepiece.

    • Resolving power represents the capacity of the optical system to produce


    separate images of objects very close to each other.
    Resolving power (R) = 0.61λ/ NA

    Where,
    ➢λ is wavelength of incidental light and
    ➢NA is numerical aperture of lens which is generally engraved on the body
    of the objective lens.

    • Resolving power of a standard light microscope is around 200 nm.


    Microscopy
    • The science of investigating small objects using such an instrument
    like microscope is called Microscopy.
    Use of The Microscope

    1. Place the microscope on a stable surface of a bench.


    2. Provide proper light source.
    3. Direct the path of light to pass through iris diaphragm, with proper
    intensity by setting the mirror.
    4. Put a slide on the stage in between the clips provided on stage.
    5. Revolve the nosepiece and adjust low power objective to the object
    on the slide.
    6. Look through the eyepiece and adjust with coarse adjustment.
    7. Now turn the high power objective to increase the magnification
    8. To get sharp image, adjust with fine adjustment.
    9. Put a small drop of immersion oil on the slide and turn oil
    immersion objective.
    10. Use the fine adjustment to get clarity in image.

    a. For unstained preparation:


    i. Lower the condenser.
    ii. Close the iris diaphragm.
    iii. Use concave mirror.
    iv. Focus under low power and then turn to high power.
    b. For stained preparation:
    i. Lower the condenser.
    ii. Use concave mirror.
    iii. Adjust the iris diaphragm to give maximum light.
    iv. Focus under low power, then turn to high power.

    c. For examination under oil immersion:


    i. Raise the condenser completely.
    ii. Open the iris diaphragm.
    iii.Use flat side of mirror.
    iv. Place a drop of immersion oil on the slide and focus under oil immersion
    objective.
    v. Touch the objective to the oil on slide.
    vi. Using fine adjustment, focus the object.
    Routine Care and Maintenance of a Microscope

    • A microscope is an expensive and delicate equipment.


    • It should be properly maintained for getting good results and long
    years of service.
    • Fungus and dust are the first enemies of the microscope.
    • Fungus grows on the lenses and dust scratches the mirror.
    • Following are some points to take care of the microscope:
    1. Carry the microscope by holding its arm with one hand and the
    other hand under the foot.
    2. When the microscope is not in use, put it under a plastic cover or in
    a wooden box.
    3. While working with microscope, do not pull the object slide from
    the stage without moving the oil immersion objective.
    4. It makes scratches to the objective.
    5. The microscope should be kept on stable even surface and in
    comfortable position.
    6. After use, remove the oil from the objective with the cloth dipped
    in xylol.
    7. Never use spirit for cleaning the lens.
    8. Leave oil immersion objective in position while it is not in use.
    9. Make a habit to clean all parts of microscope after a day's work
    10. Use clean tissue paper or lens paper or soft cloth for removing oil,
    but never use cotton.
    Image Formed By Bright Field Microscopy
    Other Types of Microscopy

    Dark Ground Illumination (DGI)


    • This method is used for examination of unstained living
    microorganisms e.g. Treponema pallidum.
    Phase Contrast Microscopy
    • Like DGI described above, phase contrast
    microscopy is also used for examination of
    unstained structures, most often living cells
    for assessing their functions at the level of
    organelles such as mobility, phagocytosis etc.

    Polarizing Microscope
    • This method is used for demonstration of
    birefringence e.g. amyloid, foreign body, hair
    etc.
    Fluorescent Microscope
    • This method is used for demonstration of naturally-occurring
    fluorescent material and other non-fluorescent substances or
    microorganisms after staining with some fluorescent dyes e.g.
    Mycobacterium tuberculosis, amyloid, lipids, elastic fibers etc.
    • Light source of low wavelength (UV light) for illumination is used,
    most often mercury vapour lamp or xenon gas lamp.
    Electron Microscope (EM)
    • EM is used for study of ultrastructural details of the tissues and cells.
    • For electron microscopy, tissue is fixed in 4% glutaraldehyde stored at
    4°C for 4 hours.
    • Ultrathin micro sections with thickness of 100 nm are cut with
    diamond knives.
    • There are two types of electron microscopy:
    1. Transmission electron microscopy (TEM)
    2. Scanning electron microscopy (SEM)
    ➢Transmission Electron Microscopy (TEM)

    • TEM helps visualize cell's cytoplasm and organelles.


    • For this purpose, ultrathin sections are required.
    • TEM interprets atomic rather than molecular properties of the tissue
    and gives two dimensional image of the tissue.
    ➢Scanning Electron Microscopy (SEM)

    • SEM helps in the study of cell surface.


    • In this three-dimensional image is produced.
    • The image is produced on cathode ray
    oscillograph which can also be amplified.
    • SEM can also be used for fluorescent antibody
    techniques.
    Thank You

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