Introduction to Microscopy By Dr. Nand Lal Dhomeja

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ANATOMY
INTRODUCTION TO MICROSCOPY
Learning objectives:
At the end of the lecture the student should be able to:
Learn the details of microscope and common terms used in microscopy
Identify different types of Microscopes and their functions
Identify parts of light Microscope
Discuss the working of Light Microscope
Discuss the magnification of Microscope
Identify the types of Electron Microscopes (Transmission and Scanning)
Microscope:
A microscope (from the Greek: "small" and "to look" or "see") is an instrument to see objects too small
for the naked eye.
Microscopy is any technique for producing visible images of structures or details too small to otherwise
be seen by the human eye.
Microscopy:
Magnifying power
Ability to enlarge the image
Resolving power
Ability to separate two points situated close together
This quality is known as resolution of a microscope
Types of microscope:
There are many types of microscopes.
Optical or light microscope which uses light to image the sample.
Electron microscope which uses invisible radiations
(transmission electron microscope and the scanning electron microscope)
Phase contrast microscope and differential interference microscope
Polarizing microscope
Confocal microscope
Flourescence microscope
Optical or light microscope:
The simplest optical microscope is the magnifying glass and is good to about ten times (10X)
magnification
Most common.
Optical instrument containing one or more lenses producing an enlarged image of a sample placed in
the focal plane is the compound microscope.
Compound microscope:
Has 2 systems of lenses for greater magnification.
1) Ocular or eyepiece :
lens that one looks into
Usually 10X or 15X power.
2) Objective lens, or the lens closest to the object.
Parts of compound microscope:
Body Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator or condenser: A steady light source (110 volts) used in place of a mirror.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be
rotated to easily change power.
Objective Lenses: There are 3 or 4 objective lenses on a microscope.
They always consist of 4X, 10X, 40X and 100X powers.
When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times
10X), 100X, 400X and 1000X.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage.
Diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that
is projected upward into the slide
Coarse adjustment
Large, round knob on the side of the microscope
Either move the stage, or the top part of the microscope up and down
Fine adjustment
Small, round knob on the side of the microscope.
Used to fine tune the focus after adjusting the coarse adjustment knob.
How a microscope does works?
Gather light from a tiny specimen that is close by.
Objective lens of a microscope is small.
The image is again magnified by a second lens called an eyepiece, as it is brought to your eye.
Electron microscope:
Electron microscope is a type of microscope that produces an electronically-magnified image of a
specimen for detailed observation.
The electron microscope (EM) uses a particle beam of electrons to illuminate the specimen and create a
magnified image of it.
Uses electrostatic and electromagnetic "lenses" to control the electron beam and focus it to form an
image.
Types of electron microscope:
Transmission electron microscope.(TEM)
Scanning electron microscope (SEM)
Transmission electron microscope
The original form of electron microscope, the transmission electron microscope (TEM) uses a high
voltage electron beam to create an image.
Limit of resolving power of the TEM is about 0.35 nm,
Magnifying power ranges up to 1000,000X or more.
Scanning electron microscope
Differs from TEM in that the electrons do not pass through the specimen under examination.
A beam is made to scan the sample surface.
Electrons reflected from the surface are collected by a detector and processed so that they can be
displayed as a three dimensional image on a tv screen.
Resolving power: 10 nm
Preparation of tissue for light microscopy
Fixation.
It is a chemical process by which biological tissues are preserved from decay, either through autolysis or
putrefaction.
Terminates any ongoing biochemical reactions
Increase the mechanical strength or stability of the treated tissues.
Most commonly used fixative is formalin
Preparation of tissue for light microscopy
Embedding
After fixation to make the tissue hard , it is embedded to obtain a hard consistency that permits it to be
sectioned.
Paraffin wax most frequently used.
Sectioning
Done with a help of machine called microtome.
Preparation of tissue for light microscopy:
Staining
Coloring the colorless substances.
To enhance natural contrast
To permits distinction between tissue components
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