Detailed summary of the microscope, how it works, care, components, and properties. It also explains the preparations of specimens for visualization under the microscope, simple staining, differential staining and special staining techniques. Further including, gram-negative and positive staining, ...
Intended Learning Outcomes (ILOS)
• Compare and contrast the features of simple and compound microscopes
• Identify and describe the parts of a brightfield microscope
• Calculate total magnification for a compound microscope
• Describe the concept of par-focal lenses
• Define the terms magnification, resolving power, numerical aperture and limit of resolution
Introduction to the Light Microscope
→ Microscope is an optical instrument that uses lens
or combination of lens to produce magnified
images that are too small to seen by unaided eye.
→ Microscope provides the enlarged view that helps
in examining and analyzing the image.
→ Microscope can be separated into optical theory
microscopes (Light microscope), electron
microscopes (eg. TEM, SEM) and scanning probe
microscopes.(eg. AFM, PSTM).
→ Optical microscopes function on the basis of
optical theory of lenses by which it can magnifies
the image obtained by the movement of a wave
through the sample.
→ The waves used in optical microscopes are
electromagnetic and that in electron microscopes
are electron beams.
→ Light microscopes can be classified into Bright
field microscope, Phase contrast microscope, Dark
field microscope and Fluorescence microscope.
Light Microscopy
Light microscope uses the properties of light to produce an enlarged image. It is the simplest type of
microscope.
Based on the simplicity of the microscope it may be categorized into:
A) Simple microscope.
B) Compound microscope.
A) Simple microscope
→ It uses only a single lens, e.g.: hand lens. Most of these are double convex or planoconvex lens.
→ The developments of advanced techniques for grinding and shaping lenses allowed professionals such as
Hans Janssen and Anton van Leeuwenhoek to develop simple microscopes which advanced the study of
biology significantly.
→ B) Compound microscope
→ In the compound microscope used two lenses or lens systems.
→ One of the lens system formed an enlarged image of the object and the second lens system magnifies the
image formed by the first.
→ The modern compound microscope consists of two lens system, the objective and the ocular or eye piece.
→ The first magnified image obtained with objective lens, is again magnified by the eye piece to give a virtual
inverted image.
→ The total magnification the product of the magnifications of two lens systems. TJW Notes
, Parts of the Microscope
It consists of mainly three parts:
1. Mechanical part - base, c-shaped arm and stage.
2. Magnifying part - objective lens and ocular lens.
3. Illuminating part - sub stage condenser, iris diaphragm, light source.
1. Mechanical part
• Base: It helps in holding the various parts of microscope. It also contains the light source.
• C-shaped arm: It is used for holding the microscope. And which is connected the eyepiece to the objective
lens.
• Mechanical stage: It is a rigid platform on which specimen to be viewed is placed. It has an aperture at the
centre to permit light to reach the object from the bottom. The object on the slide can be moved either
sideways or forward and backward with the help of the positioning knobs.
2. Magnifying part
• Eyepiece (Ocular lens):
It is the lens where the final image of the object is viewed. Usually; these lenses have a magnification of
either 10X or 15X.
• Objective lens:
There are three types of objective lens:
– 4X (scanning objective)
– 10X (Low power objective lens).
– 40X (High power objective lens).
– 100X (Oil immersion objective lens).
→ Each objective lens is represented by a particular colour.
→ Here we represents 4X with red band, 10X with yellow, 40X with blue and 100X with white.
→ These objective lenses are fitted on to the revolving nose piece.
→ The working distance of an objective is defined as the distance between the front surface of the lens and
the cover glass surface or the specimen when it is in sharp focus.
3. Illuminating part
• Sub stage condenser: It is seen below the stage and made
up of a system of convex lenses which focus light from illuminating sources
and is used to condense light towards the object. Lowering the condenser
diminishes illumination whereas raising the condenser increases the illumination.
• Iris diaphragm: It is seen immediately below the condenser and operated by
small lenses which protrude to one side. Opening and closing of iris diaphragm
controls the light reaching the object.
• Light source: Light source is situated at the base of the microscope. It is controlled
by an ON /OFF switch and a lamp rheostat. Tungsten-halogen lamps are highly
reliable light source used in the light microscope. It generates a continuous distribution of light across the
visible spectrum.
Adjustments Knobs in the Microscope
• Coarse Adjustment Knob:
objective lenses can be moved towards or away from the specimen by using this coarse adjustment knob
• Fine Adjustment Knob:
It is used to fine tune the focus on the specimen and also used to focus on various parts of the specimen.
commonly one uses the coarse focus first to get close and moves to the fine focus knob for fine tuning.
TJW Notes
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