Lecture 4
Learning objective: Two lectures will be devoted to learn about the microscopes used to observe, study, and
characterize microbes. In addition to the hands on training on how to operate microscopes, you are expected to
know how microscopes magnify a object, what the principles that we need to understand while handling
microscope. Various types microscopes and their application will also be taught in this two lectures.
Microscopy Principles Resolution Numerical Aperture, Magnification Different types of Microscopes – Light
Microscope, UV, Dark Field, Phase Contrast, Fluorescence and Electron Microscopes
A microscope is an optical instrument consisting of one or more lenses in order to magnify images of
minute objects. Microscopes are of two categories.
(I) Light Microscope: Magnification is obtained by a system of optical lenses using light waves. It includes (i)
Bright field (ii) Dark field (iii) Fluorescence (iv) Phase contrast and (v) UV Microscope
(ii) Electron Microscope: A system of electromagnetic lenses and a short beam of electrons are used to
obtain magnification. It is of two types: (I) Transmission electron microscope (TEM) (ii) Scanning electron
microscope (SEM)
Light microscopes can be broadly grouped into two categories
(a) Simple microscope : It consists of only one bi-convex lens along with a stage to keep the specimen.
(b) Compound microscope : It employs two separate lens systems namely, (i) objective and (ii) ocular (eye
piece).
The compound student microscope is a bright field microscope. It consists of mechanical and optical parts.
1. Mechanical parts
These are secondary but are necessary for working of a microscope. A ‘Base’, which is horsehoe,
shaped supports the entire framework for all parts. From the base, a ‘Pillar” arises. At the top of the
pillar through an Inclination Joint’ arm or limb is attached. At the top of the pillar, a stage with a central
circular opening called ‘Stage aperture’ is fixed, with a stage clip to fix the microscopic slide. Beneath
the stage, there is one stage called ‘sub stage’ which carries the condenser. At the top of the arm, a
hollow cylindrical tube of standard diameter is attached in -line with the stage aperture, called body
tube. The body tube moves up and down by two separate arrangements called ‘ coarse adjustment’
worked with pinion head and fine adjustment worked with micrometer head. At the bottom of the body
tube an arrangement called revolving nose-piece is present for screwing different objectives. At the top
of the body tube eye- piece is fixed.
2. Optical parts
It includes mirror, condenser, objective and ocular lenses. All the optical parts should be kept in perfect
optical axis.
a. Objectives : Usually 3 types of magnifying lenses (i) Low power objective (10x) (ii) High dry objective (45x) and
(iii) Oil immersion objective (100x)
b. Eye-piece : Mostly have standard dimensions and made with different power lenses. (5x, 10x, 15x, 20x). A
compound microscope with a single eyepiece is said to be monocular, and one with two eyepieces is said to be
binocular.
c. Condenser : Condenses the light waves into a pencil shaped cone there by preventing the escape of light waves.
Also raising or lowering the condenser can control light intensity. To the condenser, iris diaphragm is attached
which helps in regulating the light.
d. Mirror : It is mounted on a frame and attached to
the pillar in a manner that it can be focused in three
different directions. The mirror is made of a lens with
one plane surface and another concave surface. Plane
surface is used, when the microscope is with a
condenser.
Principles of Microscopy
Basically, a light microscope magnifies small objects
and makes them visible. The science of microscopy is
based on the following concepts and principles:
Magnification
Magnification is simply the enlargement of the
specimen.
The magnifying power of a compound light
microscope is the product of the individual magnifying powers of the ocular lens and the objective lens.
Ocular
Objective lens
Magnification
10X
10X (low power)
100X
10X
45X (high “dry”)
450X
10X
100X
immersion)
(oil
1000X
Magnification beyond the resolving power will result in larger
images but less distinct in their details and appearance.
Resolving power of the light microscope:
Resolution is a measure of how clearly details can be seen and
is distinct from magnification which depends on the ability of a lens.
The usefulness of the microscope depends on the resolving power,
which is the basic limitation of the bright field microscope. Resolving
power is defined as the ability to distinguish two adjacent points as
distinct and separate. The resolving power of microscope is the function of wavelength of light used and the
numerical aperture (NA) of the objective lenses. Magnification beyond the resolving power is of no value.
Numerical aperture is a measure of the ability of the objective lens to capture light.
NA = n sin θ
where, n = Refractive index of the material between the specimen and the lens.
sin θ = Half the value of cone of light that enters through the objective
Therefore. Resolving power =
-----2xNA
where = wavelength of light used
It implies that,
- Higher the NA , higher will be the degree of resolving power
- Shorter the wavelength of light, higher will be the resolving power.
For a low power objective with an NA of 0.25, the resolving power is calculated as
Resolving power = 550 nm
=
550
----------------- = 1100 nm or 1.1 µm
2 x 0.25
0.50
The resolving power of the lens system is 1.1 µm, hence any object smaller than 1.1µm could not be seen.
Contrast is the ability to distinguish an object from its background. Since most microbes are relatively transparent
when viewed under a standard light microscope they are difficult to identify. Using a stain (labs 2-5) that will bind
to the microorganism and not the glass slide, dramatically enhances their contrast enabling them to be observed
more clearly.
Depth-of-focus is the “thickness” of the sample that appears in focus at a particular
magnification. As the magnification increases the depth-of-focus decreases, or the “slice” of the sample that
appears in focus gets thinner. Many of the newer compound microscopes are par focal, which means that if one
objective lens has the object in focus, and you go to the next objective lens, only minor adjustment (fine focus) is
needed to bring the image back into focus. This is due to the fact that as you increase the magnification, and thus
the slice of the sample that appears in focus becomes “thinner”, the correct plane-of-focus will always be within
the depth of focus of the previous objective. After you get the sample into focus at scanning or low power using
the course adjustment knob, you should only have to use the fine focus knob at the higher
magnifications.
Field-of-View is the area of the slide that you are observing through the microscope. As you increase the
magnification the actual area of the slide that you are looking at is getting smaller. You can think of the field-ofview as a dartboard. At low magnification you are able to see the entire dartboard, but as you increase the
magnification you are only observing the bulls-eye, a much smaller portion of the dartboard.
These microscopes are also par central, which refers to the ability to keep an object in the middle of your field-ofview when changing from one objective to another. It is useful to remember this as you are increasing
magnification. Always keeping your sample in the center of your field-of-view will avoid unnecessary “searching”
of the slide for your sample.
Working distance is the distance between the objective and the slide. As you increase
magnification (by using more powerful objective lenses) the working distance decreases. So much so that by the
time you are using the oil-immersion objective (100X) the objective is almost touching the slide, allowing the
immersion oil to “connect” the slide and objective. It is important to consider working distance in a number of
applications, but practically there are two reasons you should be aware of your working distance. The first is so
that you do not inadvertently push the objective through the slide, causing damage to the objective and your
sample slide. The second is to estimate whether you are in the correct plane-of-focus.
Microscopy with Oil Immersion
When light passes from a material of one refractive index to material of another, as from glass to air or
from air to glass, it bends. Light of different wavelength bends at different angles so that as objects are magnified
the images become less and less distinct. This loss of resolution becomes very apparent at magnifications of above
400 x or so. Even at 400X the images of very small objects are badly distorted. The refractive index of air is 1.0,
which is less than that of glass (1.56). When the light rays passes from denser to lighter medium, the rays get
refracted. Better resolution can be obtained by using liquid medium oil, whose refractive index (1.51) is almost
equal to glass. The oil generally used is ‘cedar wood oil’. Placing a drop of oil with the same refractive index as
glass between the cover slip and objective lens eliminates two refractive surfaces and considerably enhances
resolution, so that magnifications of 1000x or greater can be achieved.
Wor k in g wi th l ig h t m ic r o sco p e : Illumination :
Adjust the concave mirror, so that the brightly illuminated microscopic field is seen through the objective.
In case of electrical source of illumination, plug the microscope in and turn on the illuminator.
Rotate the Iris diaphragm on the underside of the stage in order to adjust the light intensity. Look through
the microscope as you adjust the diaphragm and observe the changes. Set the diaphragm and move on.
Keeping the light intensity in mind, look through the microscope and move through the different
magnifications slowly. Observe the change in light intensity.
Adjust the intensity of light to match the requirements of the objective you are wishing to view.
Focusing on low power:
Keep the slide with specimen side up over the hole in the centre of the stage with the stage adjustments.
Position the 10X objective lens into the optical path, making sure that the lens clicks into position.
Look at the microscope with your head to the side. Locate the coarse adjustment knob which moves the
stage up and down. Practice moving the coarse adjustment knob, seeing how it moves the stage with
each turn of the knob. Using the coarse adjustment knob, move the stage down as far as it will go. This is
the starting position when you first begin to bring an object into focus. Look into the eyepiece. Slowly,
move the stage up by using the coarse adjustment knob until it comes into focus. Never allow the slide to
come in contact with the objective lens.
Turn the fine adjustment knob to bring the specimen into sharp focus.
You may need to change the aperture (opening) of the iris diaphragm to control the brightness. It
depends on the specimen and the magnification you are using.
Moving to a higher magnification -Do not touch the slide with the lens.
Rotate the revolving nosepiece to position 40X objective into the optical path. Use ONLY the fine
adjustment knob to bring the image into sharp focus.
Similarly rotate the revolving nose piece to position oil immersion objective. Place a drop of immersion oil
over the slide portion. While looking from the side, lower the objective such that it touches the oil and not
the slide. Perceive the image by moving the stage back and froth and focus the image accurately using
fine adjustment. Adjust the light intensity and iris diaphragm for optimum illumination
After use, clean the oil with lens paper.
NEVER use the coarse adjustment when focusing on the high dry power 40Xor Oil immersion (100X) objectives!!!!!
Care of Microscopes:
Microscopes are very expensive pieces of scientific equipment and must be treated with care. Each group of
students will be assigned a microscope to use throughout the semester. Some basic rules of microscope care
include the following:
1. Always carry a microscope with two hands, one on the base and one on the arm.
2. Use the coarse focus knob on the lowest objective only. NEVER use coarse focus on high power or oil
immersion, or you may damage the objective lenses.
3. Always clean all lenses thoroughly with lens paper and lens cleaner before putting away. Immersion oil
which is not removed immediately can dry on the lenses, making it difficult to view any specimen. Dried
immersion oil is also quite difficult to remove from the lenses.
4. Always store the microscope with the lowest objective (4X) in place. NEVER store the microscope with the
oil immersion objective in place, as it can damage the lens.