Microscopy :

Principles and description of light and electron

Microscope

A K GUPTA
 INTRODUCTION
 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.
• In 1673, Antony van Leeuwenhoek invented
microscope consisting of a biconcave lens enclosed in
two metal plates,
• Over the years, microscopes have evolved from the
simple, single-lens instrument of Leeuwenhoek, with a
magnification of 300, to the present-day electron
microscopes capable of magnifications greater than
250,000.
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Microscopes are of two types :
A.) Light microscopes ( use visible light or ultraviolet rays
to illuminate specimens)
It include: Bright-field , Dark-field Phase-contrast and
Fluorescent instruments ( use ultraviolet radiations whose
wavelengths are shorter than those of visible
light and are not directly perceptible to the
human eye) or
B.) Electron microscopes (use electron beams instead of
light rays, and magnets instead of lenses to
observe submicroscopic particles.) 3
Brightfield Microscope :
 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:
1.) Simple microscope.
2.) Compound microscope.
  1.) Simple microscope 
It uses only a single lens, e.g. hand lens.
Most of these are double convex.

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2.) Compound microscope :
• In the compound microscope, it is used two lenses or lens systems:
the ocular lens in the eyepiece and the objective lens located in
the nose-piece.
• The specimen is illuminated by a beam of tungsten light focused
on it by a sub-stage lens called a condenser, and the result is that
the specimen appears dark against a bright background.
• 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 total magnification  the  product of the magnifications of 
two lens systems. 
• A major limitation of this system is the absence of contrast
between the specimen and the surrounding medium, which
makes it difficult to observe living cells. Therefore, most bright-
field observations are performed on nonviable, stained
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preparations. 
Dark-field Microscope :
• This is similar to the ordinary light microscope;
however, the condenser system is modified so that
the specimen is not illuminated directly.
• The condenser directs the light obliquely so that the
light is scattered from the specimen, which then
appears bright against a dark background.
• Living specimens may be observed more readily
with dark-field than with bright-field microscopy. 
Phase-Contrast Microscope
• Observation of microorganisms in an unstained state is possible with
this microscope.
• Its optics include special objectives and a condenser that make
visible cellular components that differ only slightly in their refractive
indexes.
• As light is transmitted through a specimen with a refractive index
different from that of the surrounding medium, a portion of the light
is refracted (bent) due to slight variations in density and thickness of
the cellular components.
• The special optics convert the difference between transmitted light
and refracted rays, resulting in a significant variation in the intensity
of light and thereby producing a discernible image of the structure
under study. The image appears dark against a light background.
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Fluorescent Microscope :
• This microscope is used most frequently to visualize specimens
that are chemically tagged with a fluorescent dye.
• The source of illumination is an ultraviolet (UV) light obtained from
a high-pressure mercury lamp or hydrogen quartz lamp.
• The ocular lens is fitted with a filter that permits the longer
ultraviolet wavelengths to pass, while the shorter wavelengths are
blocked or eliminated.
• Ultraviolet radiations are absorbed by the fluorescent label and
the energy is reemitted in the form of a different wavelength in the
visible light range.
• The fluorescent dyes absorb at wavelengths between 230 and 350
nanometers (nm) and emit orange, yellow, or greenish light.
• This microscope is used primarily for the detection of antigen-
antibody reactions. 8
 B.) Electron Microscope :
• This instrument provides with
magnifications up to one
million.
• This permits visualization of
submicroscopic cellular
particles as well as viral
agents.
• In the electron microscope,
the specimen is illuminated
by a beam of electrons rather
than light, and the focusing is
carried out by electromagnets
instead of a set of optics. 9
•These components are sealed in a tube in which a complete
vacuum is established.
• Transmission electron microscopes require specimens that
are thinly prepared, fixed, and dehydrated for the electron
beam to pass freely through them.
• As the electrons pass through the specimen, images are
formed by directing the electrons onto photographic film,
thus making internal cellular structures visible.
•Electron microscopes are used for visualizing a three-
dimensional image of surface and intracellular structures.

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 Intro to Electron Microscopy

• Similar to optical microscopy except with electrons rather than

photons
• Used to image samples with a resolution of 10 Å
– Can image many different structural geometries
• Mostly limited by radiation damage from the electron beam
 Microscope Setup

• Transmission Electron
Microscope
– Phase contrast Image is
formed by the interference
between electrons that
passed through the sample
and ones that did not
• Scanning Electron Microscope
– Electron beam is scanned
across the sample
– The reemitted electrons
are measured in order to
form the image
Theoretical Principles of Microscopy 
To use the microscope efficiently and with minimal
frustration, you should understand the basic principles of
microscopy: magnification, resolution, numerical
aperture, illumination, and focusing. 
Magnification
Enlargement or magnification of a specimen is the
function of a two-lens system; the ocular lens is found in
the eyepiece, and the objective lens is situated in a
revolving nose-piece. These lenses are separated by the
body tube. The objective lens is nearer the specimen and
magnifies it, producing the real image that is projected up
into the focal plane and then magnified by the ocular lens13
The actual power or magnification of a compound optical
microscope is the product of the powers of the ocular
(eyepiece) and the objective lens. The maximum normal
magnifications of the ocular and objective are 10× and
100× respectively, giving a final magnification of 1,000×.
 
The most commonly used microscopes are equipped
with a revolving nosepiece containing four objective lenses
possessing different degrees of magnification. When these
are combined with the magnification of the ocular lens, the
total or overall linear magnification of the specimen is
obtained. 
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Resolving Power or Resolution
Although magnification is important, unlimited enlargement is
not possible by merely increasing the magnifying power of the
lenses or by using additional lenses, because lenses are limited
by a property called resolving power.
By definition, resolving power is the ability of a lens to show
two adjacent objects as discrete entities.
• When a lens cannot discriminate, that is, when the two objects
appear as one, it has lost resolution.
• Increased magnification will not rectify the loss, and will, in
fact, blur the object.
• The resolving power of a lens is dependent on the wave-length
of light used and the numerical aperture, which is a
characteristic of each lens and imprinted on each objective. 15
 The numerical aperture is defined as a function of the diameter of the
objective lens in relation to its focal length. It is doubled by use of the
sub stage condenser; which illuminates the object with rays of light
that pass through the specimen obliquely as well as directly.
Numerical Aperture (NA)= ŋsinƟ
Where, ŋ is refractive index of the medium ( Refractive index of air is 1
and of oil immersion is 1.56).
And Ɵ = The angle subtended by the optical axis and the outermost rays
still covered by the objective is the measure of the aperture of the
objective, it is the half aperture angle. ( For low power objective total
angle=64 degree so Ɵ = 32 degree and same as 48 degree and 58
degree for high power and oil immersion respectively)
For e.g. NA= ŋsinƟ = 1.56 x sin 58 =1.56 x 0.85 = 1.33 ( for oil
immersion)
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Thus, resolving power is expressed mathematically, as
follows: 
Resolving power = Wavelength of Light/ 2 (Numerical
Aperture) 

The numerical aperture of a microscope objective is

a measure of its ability to gather light and resolve fine
specimen detail at a fixed object distance. Image-forming
light waves pass through the specimen and enter the
objective in an inverted cone as illustrated in Figure 1.
A longitudinal slice of this cone of light shows the
angular aperture, a value that is determined by the
focal length of the objective. 17
Based on this formula,
• the shorter the wave-length, the greater the resolving
power of the lens. (Thus, short wavelengths of the
electromagnetic spectrum are better suited than longer
wavelengths in terms of the numerical aperture.)
• visible portion of the electromagnetic spectrum is very
narrow and borders on the very short wavelengths found
in the ultraviolet portion of the spectrum
• The relationship between wavelength and numerical
aperture is valid only for increased resolving power when
light rays are parallel.
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• Therefore, the resolving power is dependent on another factor, the
refractive index. This is the bending power of light passing through
air from the glass slide to the objective lens.
• The refractive index of air is lower than that of glass, and as light
rays pass from the glass slide into the air, they are bent or refracted
so that they do not pass into the objective lens.
• This would cause a loss of light, which would reduce the numerical
aperture and diminish the resolving power of the objective lens.
• Loss of refracted light can be compensated for by interposing
mineral oil, which has the same refractive index as glass, between
the slide and the objective lens.
• In this way, decreased light refraction occurs and more light rays
enter directly into the objective lens, producing a vivid image with
high resolution. 19
 Illumination
Effective illumination is required for efficient magnification
and resolving power. Since the intensity of daylight is an
uncontrolled variable, artificial light from a tungsten
lamp is the most commonly used light source in
microscopy.
• The light is passed through the condenser located
beneath the stage.
• The condenser contains two lenses that are necessary to
produce a maximum numerical aperture. The height of
the condenser can be adjusted with the condenser knob.
• Always keep the condenser close to the stage, especially
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when using the oil-immersion objective. 
• Between the light source and the condenser is the iris
diaphragm, which can be opened and closed by means
of a lever; thereby regulating the amount of light
entering the condenser.
• Excessive illumination may actually obscure the
specimen because of lack of contrast.
• The amount of light entering the microscope differs with
each objective lens used. A rule of thumb is that as the
magnification of the lens increases, the distance
between the objective lens and slide, called working
distance, decreases, whereas the numerical aperture of
the objective lens increases. 21
 Parts of a 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.
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 22
knobs.
Structural' components numbered below
according to the image on the left :

1. Eyepiece (ocular lens)

2. Objective turret, revolver, or revolving
nose piece (to hold multiple objective
lenses)
3. Objetive nobes
Focus knobs (to move the stage)
4. Coarse adjustment
5. Fine adjustment
6. Stage (to hold the specimen)
7. Light source (a light or a  mirror)
8. Diaphragm and condenser 
9. Mechanical stage

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Image of a Compound microscope

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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 color. 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
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the cover glass surface  or the specimen when it is in sharp focus.
Illuminating part 
1. 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.
2. 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. 26
3. 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 
a) Coarse Adjustment Knob:
objective lenses can be moved towards or away from the
specimen by using this coarse adjustment knob
b) 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.
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Focusing On Microscopic Objects: 
Start with Clean Lenses:
It is important that microscope lenses be very clean. Before viewing
through a microscope, use lens paper to gently clean the lenses.
 
Begin at Low Power Magnification: 
Always begin by viewing the object through a low power lens. Depending
on how small the object is, start with the scanning or low-power objective.
 
Using low-power objective lens, get the target object centered in the field-
of-view and focus as much as possible, first by using the coarse focus and
then fine-tuning the clarity of the image with the fine focus.
 
Once the object is in focus, switch to the next higher objective power. Do
not change the focus or manipulate the focus knobs in any way while
changing objectives.

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Adjustments for oil immersion objective: 
Without changing the adjustment of high power, turn to oil immersion
objective. One drop of oil is  added  into on the slide. The nose piece is
turned such that the oil immersion objective touches on the drop of oil.
Open the iris diaphragm completely. Use only fine adjustments for
focusing.
 
After changing to a higher objective (such as high-dry or oil-immersion)
the viewer needs only manipulate the fine focus knob. Never manipulate
the coarse focus at oil immersion. Manipulating the coarse focus at high
power can smash the lens into the slide, potentially damaging the scope
and the specimen.

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Use and Care of the Microscope
 
You will be responsible for the proper care and use of microscopes. Since
microscopes are expensive, you must observe the following regulations
and procedures. 

• The instruments are housed in special cabinets and must be moved by

users to their laboratory benches.

• The correct and only acceptable way to do this is to grip the microscope
arm firmly with the right hand and the base with the left hand, and lift the
instrument from the cabinet shelf.

• Carry it close to the body and gently place it on the laboratory bench. This
will prevent collision with furniture or co-workers and will protect the
instrument against damage.

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 Once the microscope is placed on the laboratory bench, observe the following
rules:
1. Remove all unnecessary materials such as books, papers, purses, and hats from
the laboratory bench.
2. Uncoil the microscope's electric wire and plug it into an electrical outlet.
3. Hold the plug (not the cable) when unplugging the illuminator.
4. lean all lens svstems; the smallest bit of dust, oil, lint, or eyelash will decrease
the efficiency of the microscope.
• The ocular; scanning, low-power, and high-power lenses may be cleaned by
wiping several times with acceptable lens tissue.
• Never use paper toweling or cloth on a lens surface.
• If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened
with methanol is used to wipe it clean.
• If the lens is very dirty it may be cleaned with xylol however the xylol
cleansing procedure should be performed only by the instructor, and only if
necessary. Consistent use of xylol may loosen the lens.
• Since bulbs are expensive, and have a limited life, turn the illuminator off when
you are done.

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The following routine procedures must be followed to ensure correct and
efficient use of the microscope while focusing. 
1. Place the microscope slide with the specimen within the stage clips on
the fixed stage. Move the slide to center the specimen over the opening
in the stage directly over the light source.
2. Rotate the scanning lens or the low power lens into position. While
watching from the side to insure that the lens doesn't touch the
specimen, turn the coarse focus knob to move the stage as close as it can
get to the lens without touching the lens. (Always watch from the side
whenever you move a specimen towards any objective lens to make sure
the lens doesn't crash through the specimen and get damaged!)
3. Now, while looking through the ocular lens, turn the coarse focus knob
carefully, and slowly move the stage away from the lens until the
specimen comes into vague focus. Then, use the fine focus knob to bring
the specimen into sharp focus.
5. Routinely adjust the light source by means of the light source transformer
setting, and/or the iris diaphragm, for optimum illumination for each new
slide and for each change in magnification.

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6. Once you have brought the specimen into sharp focus with a low-
powered lens, preparation may be made for visualizing the specimen
under oil immersion. Place a drop of oil on the slide directly over the area
to be viewed. Rotate the nosepiece until the oil-immersion objective
locks into position. Care should be taken not to allow the high-power
objective to touch the drop of oil. The slide is observed from the side as
the objective is rotated slowly into position. This will ensure that the
objective will be properly immersed in the oil. The fine-adjustment knob
is readjusted to bring the image into sharp focus
7. 8. During microscopic examination of microbial organisms, it is always
necessary to observe several areas of the preparation. This is
accomplished by scanning the slide without the application of additional
immersion oil. This will require continuous, very fine adjustments by the
slow, back-and-forth rotation of the fine adjustment knob only.

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 On completion of the laboratory exercise, return the microscope to its
cabinet in its original condition. The following steps are recommended:
1. Since bulbs are expensive, and have a limited life, turn the illuminator off
when you are done.
2. Clean all lenses with dry, clean lens paper. If you need to, you can use a
drop or two of methanol to help clean the lens. Use xylol to remove oil
from the stage only.
3. Place the low-power objective in position and bring the stage and
objectives close together.
4. Center the mechanical stage.
5. Coil the electric wire around the body tube and the stage.
6. Carry the microscope to its position in its cabinet in the manner
previously described.
7. Cover the instrument with a dust jacket when not in use.
8. Focus smoothly; don't try to speed through the focusing process or force
anything. For example if you encounter increased resistance when
focusing then you've probably reached a limit and you are going in the
wrong direction. 34
Thank You

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