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Microscope is an optical instrument that uses a lens or combination of lenses to produce magnified images
that are too small to be seen by an unaided eye. Microscope provides the enlarged view that helps in
examining and analyzing the image. Microscopes 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 magnify 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 microscopes, Phase contrast microscope, Dark field microscope and Fluorescence
microscope.

Parts of a 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 - substage condenser, iris diaphragm, light source.

OBJECTIVES:

In this module , we are going to :

1. Identify the parts and function of a compound light microscope;

2. Understand common microscopy terms;
3. Calculate microscope magnification setting; and
4. Explore virtual microscopy laboratory as a simulation experience.

MATERIAL

Any opaque object Old newspaper

Microscope Slides with mounted letters
scissor
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Light Microscopy

Light microscopy 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 plano convex 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

The compound microscope used two lenses or lens systems. One of the lens systems 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 systems, the objective and the ocular or eyepiece. The first
magnified image obtained with an objective lens, is again magnified by the eyepiece to give a virtual
inverted image. The total magnification is the product of the magnifications of two lens systems.

In this type of microscope, there are ocular lenses in the binocular eyepieces and objective lenses in a
rotating nosepiece closer to the specimen. Because It contains its own light source in its base, a
compound light microscope is also considered a bright field microscope. Bright field microscopy simply
means that the specimen is lit from below and viewed from above.
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Although sometimes found as monocular with one ocular lens, the compound binocular microscope is
more commonly used today. Microscopes have come a long way since then—today's strongest compound
microscopes have magnifying powers of 1,000to 2,000X.

Identify the parts of a microscope:

Magnification

In order to ascertain the total magnification when viewing an image with a compound light microscope, take
the power of the objective lens which is at 4x, 10x or 40x and multiply it by the power of the eyepiece which
is typically 10x.

Therefore, a 10x eyepiece used with a 40X objective lens, will produce a magnification of 400X. The
naked eye can now view the specimen at a magnification 400 times greater and so microscopic details
are revealed.

Magnification is the ability to view an object as larger. A good image is obtained when the amount
of specimen detail is also increased. Magnification alone will not achieve this.
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Resolution

Good resolution or the resolving power of the microscope is necessary to see the valuable details in an
image. Resolving Power is the ability to measure the separation of images that are close together.

Working Distance

At low magnification your working distance is longer and so vice versa when increasing magnification.

Damage to your specimen is inevitable if you are not cautious of the shorter working distance
when increasing your magnification.

Be especially careful with oil immersion lenses. This objective has the smallest working distance and
your careful handling is important.

Parts and Function of a Microscope

Parts of a Compound Microscope Function

LENS the viewer looks through to see the specimen. The eyepiece usually
contains a 10X or 15X power lens.

Diopter Adjustment Useful as a means to change focus on one eyepiece so as to correct for
any difference in vision between your two eyes.

Body tube (Head) The body tube connects the eyepiece to the objective lenses.

Arm The arm connects the body tube to the base of the microscope.

Coarse adjustment Brings the specimen into general focus.

Fine adjustment Fine tunes the focus and increases the detail of the specimen.

Nosepiece A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses.

Objective lenses A standard microscope has three, four, or five objective lenses that range
in power from 4X to100X.

Stage The flat platform where the slide is placed.

Stage clips Metal clips that hold the slide in place.

Stage height adjustment These knobs move the stage left and right or up and down.
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(Stage Control)

Aperture The hole in the middle of the stage that allows light from the illuminator
to reach the specimen.

Illumination The light source for a microscope. Older microscopes used mirrors to
reflect light from an external source up through the bottom of the stage;
however, most microscopes now use a low-voltage bulb.

Iris diaphragm Adjusts the amount of light that reaches the specimen.

Condenser Gathers and focuses light from the illuminator onto the specimen being
viewed.

Base The base supports the microscope and it’s

where the illuminator is located.

Image Formation

The direct or undeviated light from a specimen is projected by the

objective and it spreads evenly across the entire image plane at the
diaphragm of the eyepiece. The light diffracted by the specimen has
come to focus at different localized sites on the same image plane,
and the diffracted light causes destructive interference. One of the
consequences is the reduction in light intensity resulting in greater or
lesser dark areas. The patterns of light and dark that are recognized
as an image of the specimen. Because our eyes are very sensitive to
variations in brightness, and then the image becomes a more or less
faithful reconstitution of the original specimen.

The objective lens at first formed a real and inverted magnified image. And then the eye piece further
magnifies the same image to a virtual magnified image.
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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 a 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.

Adjustments for oil immersion objective:

Without changing the adjustment of high power, turn to oil immersion objectives. One drop of oil is
added onto 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.

The Importance of Par focal:

A set of objectives on a microscope are said to be par focal if the viewer can change from one to another
and still have the specimen nearly in focus. This is a very convenient feature, because as the
magnification increases, even small manipulations of the focus knob can take a specimen far out of
focus.
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.

Key Points
Magnification: Magnification is defined as the degree of enlargement of an object provided by the microscope.
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Magnification of a microscope is the product of the individual magnifying ability of an ocular lens and objective
lens.

Magnification of ocular lens Magnification of objective lens Total magnification

10X 4X 40X
10X 10X 100X
10X 40X 400X
10X 100X 1000X

Resolving power:
It is defined as an ability to distinguish between two particles situated very close.

Numerical aperture:
It is defined as the property of the lens that decides the quantity of light that can enter. The angle of the
cone of light entering an objective is known as theta
NA=nsinθ ; NA=numerical aperture

n=refractive index of the imaging medium between the front lens of the objective and the specimen cover
glass, a value that ranges from 1.00 for air to 1.51 for specialized immersion oils.

θ= one-half of the angular aperture (A)

Resolution is a subjective value in microscopy because at high magnification, an image
may appear not very sharp but still it can be resolved to the maximum ability of the
objective. Numerical aperture is defined as the resolving power of lens objective, but
the entire resolution of a microscope system also depends on the numerical aperture
of the substrate condenser. For getting a better resolution, higher the numerical
aperture of the total system.
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Properties of Microscopic Objective Lens

Property Scannin Low High Oil

g Power Power Immersion
Magnification 4x 10x 40-45x 90-100x
Numerical Aperture 0.10 0.25 0.55-0.65 1.25-1.4
Approximate focal length 40 mm 16mm 4 mm 1.8-2.0 mm
Working distance 17-20 4-8 mm 0.5-0.7 mm 0.1 mm
mm
Approximate resolving power with light of 450 nm(blue 2.3 μm 0.9 μm 0.35 μm 0.18 μm
light)

Step-by-Step Instructions on how to use and adjust a compound microscope:

1. Capture the source of light. You may turn on the illuminator or adjust the mirror to catch light. When
using the dimmer, it is best to slowly increase the light intensity as the lamp heats up quite quickly.

2. Place a slide or specimen on the stage with the sample directly above the aperture and, if possible,
fasten it to the stage with the stage clips. Reminder: A cover slip is always needed to allow for the best
quality image.
3. Ensure the iris diaphragm is completely open, allowing the maximum amount of light to reach the slide
and the lenses. Caution: Do not use the iris diaphragm to control the light, it is to control resolution and
contrast - use the dimmer instead.

4. Rotate the nosepiece so that the objective lens with the lowest level of magnification is directly above
the sample. Reminder: Using lower magnification first helps to select the part of the specimen of interest
and then adjust further.

5. Look through the binocular eyepieces and adjust the iris diaphragm until the amount of light is
satisfactory. More light is better than less light, but the comfort of the viewer’s eyes should also be taken
into account.

6. Turn the coarse adjustment knob until the specimen comes into broad focus. Caution: you should not
use the coarse focus with a high magnification objective for fear of the objective making contact with the
slide.
7. Turn the fine adjustment knob until the specimen comes into sharp focus. Caution: should not take a
long time to find focus, otherwise the high magnification objective could also hit the slide. If you are having
a difficult time to find focus then restart with the lower magnification objective.

8. The viewer should then be able to rotate the nosepiece to higher settings and bring the sample into
more and more detail with a minimal amount of refocusing.
9. In order to move your microscope safely, one hand should be under its base for support and the other
at its arm. Be sure to only switch off the microscope when the dimmer is set to the lowest intensity and
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always turn off the lamp before moving the microscope.

Best Practices in cleaning the Microscope

To best protect your microscope you need to make sure your working environment is a place where there
would be less environmental contamination with a constant temperature (little humidity) and that your
microscope sits on a solid workstation to decrease vibration.

Care of the Microscope

The microscope is a delicate instrument that should be properly used. Fungal growth on the lens or
scratches caused by dust can ruin the lenses. So the microscope should be handled carefully.

1. Carry the microscope by holding the C-shaped arm with one hand and other hand under the base. Never swing
the microscope while carrying.
2. Never allow direct light to fall on the microscope. Cover the microscope with a plastic cover when not in use.
3. While using an oil immersion objective, do not adjust the coarse screw.

4. Oil immersion objectives should be cleaned after use by wiping with soft cotton cloth or lens paper.

5. Dry objects should never come in contact with oil.

6. At the end of every experiment, clean the lenses with lens paper.

7. The scanning objective or the 4x objective should be locked in place in the revolving nosepiece, the stage should
be centered and objectives should be rolled up away from the stage, when the microscope is replaced after use.
8. When the microscope is replaced in the cabin,the microscope's arm/pillar must face the opening of the cabin.
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A. Be Careful with Solvents

First of all it is imperative that you adhere to your microscope manufacturer's recommendations and
instructions regarding the cleaning products you use when cleaning your microscope. Using a cleaning
agent that is not recommended may cause damage to the coating of optical surfaces. It is not necessary to
clean the optics after each use. If you are able to see dust and debris then imaging will be impaired so a
cleaning is necessary but keep in mind that each cleaning comes with the risk of scratching the optics. The
use of solvents is generally not recommended. Oftentimes too much solvent is used. Your microscope's
lens construction and mechanical components can be damaged by your unintentional seeping of solvents
onto these surfaces. When solvents come in contact with the cement used in lens assembly, you end up
with corrosion and separation. Another important point is the risk to your health when touching and
breathing in solvents. Liquid window cleaners and eyeglass cleaners are not meant for your microscope's
delicate optics.

Try removing surface dirt/dust with a shot of air from a squeeze bulb. Simply using distilled water is a good
and safe first choice when a cleaning agent is needed.Otherwise 90% and more pure isopropyl alcohol is
next. Place a drop or two of the alcohol on your cloth and cover and hold against the debris for a few
seconds. This will help to dissolve the culprit and then remove with a gentle wipe.

B. Immersion Oil
Oil immersion microscopy is a major culprit in that the improper cleaning and use of the immersion oil
leads to debris that attaches to the residual oil as it accumulates. Irreparable damage can occur to the
optics and mechanical components with its misuse and improper clean-up. Lens paper can be used to
remove immersion oil wiping gently/softly. Also, pay special attention to the age of the oil and its change
in viscosity over time as these can make it more difficult to remove.

C. The Optics

Never clean the internal lens surfaces. Image sharpness will be affected. This should only be done by a
qualified service center. The top portion of the eyepiece tube and the ocular rims should not be cleaned
with any moistened cloth but a dry cotton cloth instead while not touching the optics themselves. Hard
Particulates can be present on cleaning cloths that you might naturally gravitate towards to use in
cleaning your microscope. Like for instance facial tissues or paper towels, these are soft to the touch but
can leave contaminants. Special lens cloths are recommended instead.
NAME: _____________________________________ DATE:____________________________
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YEAR & SECTION: ________________________________ SCORE: __________________________

1. Draw your microscope and label at least 10 parts that are classified as structural and optical parts (10 pts.)
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2. Complete Table 1 below by supplying the characteristics of each objective. (15pts)

Table 1. Numerical characteristics of microscope objectives.

LPO HPO OIO

Focal length(mm)

Working
distance(mm)
Linear
magnification
(X)
Numerical aperture
(N.A.)
Features (color of
the band,
diameter of the
opening of the
lens, etc.)

3. Draw the position of the letter "e" as viewed under the Scanner, and the actual position of letter “e”
if you were to put it on the stage. Explain why this happens.

4. Define Parfocal. (2pts)

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5. Describe the movement of the image as seen under when:

a. Moved from side to side:

b. Moved from upward to downward:

6. Draw the image under Scanner and LPO and answer questions that follow.

a. Was there an increase or a decrease in the covered area of the specimen?

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b. Was the orientation of the letter “e” changed by the shifting from
scanner to LPO?

c. If the tail end of the letter “e” is to be viewed under LPO, where
should it (tail end) be positioned under Scanner? Describe.

d. For quick and easy focusing of the specimen, what should be the first step before shifting to
higher objectives? (5pts)

7. Compute for the total magnification with the different objectives.Table 2 below. (8pts)

Ocular Scanner 4X LPO HPO OIO

10X 40X 100X

10X
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15X

Table 2. Total magnification per objective.

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8. Microscopic Observation of Prepared Slides. Write total magnification.

Fig 1A. Plant cell (LPO) Fig 1B. Plant cell (HPO)
____X ____ X

Fig 2A. Onion root (LPO) Fig 2B. Onion root (HPO)
____X _____ X

Fig 3A. Blood (LPO) Fig 3B. Blood (HPO)

___X _____X
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9. Discuss the proper way of handling a microscope. (5pts)

10. Why should you not touch the microscope lens with your fingers? (5pts)
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11. What is an oil immersion lens? (2pts)

12. Why should you move the lenses in order as you switch from one objective to the other? (5pts)

13. What happens to the specimen as you move the magnification from 4x to 10x and 40x? (5pts)

14. How does the coarse adjustment and fine adjustment help in viewing the specimen? (3pts)
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References

(1) Light Microscope (Theory) : Cell biology Virtual Lab I : Biotechnology andBiomedical Engineering:
Amrita Vishwa Vidyapeetham Virtual Lab.vlabamritaedu. [Accessed 13 of
March]Number

(2) Reading: Microscopy | Biology (Early Release). courseslumenlearningcom.[accessed 13 of March]

https://courses.lumenlearning.com/bio1/chapter/reading-microscopy/.

(3) What is Microscopy? | Edinburgh Imaging: University of Edinburgh.[accessed

13 of March]https://www.ed.ac.uk/clinical-sciences/edinburgh-imaging/for-patients-
studyparticipants/tell-me-more-about my-scan/what-is-microscopy

(4) Bartolome FA, Quiles E. 2020. Microbiology and Parasitology. Second Edition.839 EDSA South
Triangle, Quezon City, Philippines: C&E Publishing, Inc. [accessed 13 of March