MABALACAT CITY COLLEGE

INSTITUTE OF ARTS, SCIENCES AND TEACHER

EDUCATION
(IASTE)

MODULE 1
(November 09 - 13, 2020)

BSSE 119
BIOTECHNIQUES

ROWENA V. ALDANA
Instructor
MABALACAT CITY COLLEGE
INSTITUTE OF ARTS, SCIENCES AND TEACHER
EDUCATION
 Mabalacat City, Pampanga

Module 1 – BSSE 119 - BIOTECHNIQUES

THE MICROSCOPE
Compiled and Prepared by ROWENA V. ALDANA
1st Semester A.Y. 2020-2021

I. OVERVIEW

This module deals with the understanding and discusses the different parts of the microscope that
is used to magnify small objects. Some microscopes can even be used to observe an object at the cellular
level, allowing scientists to see the shape of a cell, its nucleus, mitochondria, and other organelles.

II. LEARNING OBJECTIVES

This module is designed for you to:

 Name the parts of the microscope and their functions.

 Use the microscope to observe biological specimens
 Know the difference between the light microscope and the compound and electron microscope
 Understand the science of micrometry

III. How to complete this module?

1. Read the given module

2. Complete the ACTIVITY and EVALUATION. Due date of your accomplished worksheets will be on
November 21, 2020

3. If any questions or clarification on the lesson, leave a message in our Facebook group chat or in my Facebook
messenger https://www.facebook.com/Rowena V. Aldana or email me at [email protected], from
Saturday at 8 AM to 11 AM.

4. You can use the following link provided for additional video presentations that you can use as references.
https://www.microscopemaster.com/parts-of-a-compound-microscope.html
https://www.youtube.com/watch?v=RKA8_mif6-E&t=65s
https://www.youtube.com/watch?v=6jkK5fhZesA&t=83s
https://www.youtube.com/watch?v=pFSvM8KD5BY&t=95s
https://www.youtube.com/watch?v=1mK9EmY_r1o&t=18s
https://www.youtube.com/watch?v=erMdBKj9E3I

IV. LECTURE

WHAT IS A MICROSCOPE?
 Microscope, instrument that produces enlarged images of small objects, allowing the
observer an exceedingly close view of minute structures at a scale convenient for examination
and analysis. Although optical microscopes are the subject of this article, an image may also be
enlarged by many other wave forms, including acoustic, X-ray, or electron beam, and be
received by direct or digital imaging or by a combination of these methods. The microscope may
provide a dynamic image (as with conventional optical instruments) or one that is static (as with
conventional scanning electron microscopes).

The most familiar type of microscope is the optical, or light, microscope, in which
glass lenses are used to form the image. Optical microscopes can be simple, consisting of a
single lens, or compound, consisting of several optical components in line. The hand magnifying
glass can magnify about 3 to 20×. Single-lensed simple microscopes can magnify up to 300×—
and are capable of revealing bacteria—while compound microscopes can magnify up to 2,000×.
A simple microscope can resolve below 1 micrometer (μm; one millionth of a meter); a
compound microscope can resolve down to about 0.2 μm.

Other types of microscopes use the wave nature of various physical processes. The most
important is the electron microscope, which uses a beam of electrons in its image formation.
The transmission electron microscope (TEM) has magnifying powers of more than 1,000,000×.
TEMs form images of thin specimens, typically sections, in a near vacuum. A scanning electron
microscope (SEM), which creates a reflected image of relief in a contoured specimen, usually
has a lower resolution than a TEM but can show solid surfaces in a way that the conventional
electron microscope cannot. There are also microscopes that use lasers, sound, or X-rays.
The scanning tunneling microscope (STM), which can create images of atoms, and
the environmental scanning electron microscope (ESEM), which generates images using
electrons of specimens in a gaseous environment, use other physical effects that further extend
the types of objects that can be examined.

PARTS AND FUNCTIONS

Eyepiece: The 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: One of the most important parts of a compound microscope, as they are the
lenses closest to the specimen. 

A standard microscope has three, four, or five objective lenses that range in power from
4X to 100X. When focusing the microscope, be careful that the objective lens doesn’t touch the
slide, as it could break the slide and destroy the specimen.

Specimen or slide: The specimen is the object being examined. Most specimens are mounted on
slides, flat rectangles of thin glass.

The specimen is placed on the glass and a cover slip is placed over the specimen. This
allows the slide to be easily inserted or removed from the microscope. It also allows the
specimen to be labeled, transported, and stored without damage.

Stage: The flat platform where the slide is placed.

Stage clips: Metal clips that hold the slide in place.

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

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

On/off switch: This switch on the base of the microscope turns the illuminator off and on.

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 illuminator is located.

How Does a Compound Microscope Work?

All of the parts of a microscope work together - The light from the illuminator passes through the
aperture, through the slide, and through the objective lens, where the image of the specimen is
magnified.

The then magnified image continues up through the body tube of the microscope to the eyepiece,
which further magnifies the image the viewer then sees.

Learning to use and adjust your compound microscope is the next important step.

It's also imperative to know and understand the best practices of cleaning your microscope.

The parts of a compound microscope work together in hospitals and in forensic labs, for

scientists and students, bacteriologists and biologists so that they may view bacteria, plant and
animal cells and tissues, and various microorganisms the world over.

Compound microscopes have furthered medical research, helped to solve crimes, and they have
repeatedly proven invaluable in unlocking the secrets of the microscopic world.

Microscope Resolution: 
In microscopy, the term ‘resolution’ is used to describe the ability of a microscope to distinguish
detail. In other words, this is the minimum distance at which two distinct points of a specimen
can still be seen - either by the observer or the microscope camera - as separate entities.

Resolution and numerical aperture

The numerical aperture (NA) is related to the refractive index (n) of a medium through which
light passes as well as the angular aperture (α) of a given objective (NA= n x sin α). The
resolution of a microscope is not solely dependent on the NA of an objective, but the NA of the
whole system, taking into account the NA of the microscope condenser. More image detail will
be resolved in a microscope system in which all of the optical components are correctly aligned,
have a relatively high NA value and are working harmoniously with each other. Resolution is
also related to the wavelength of light which is used to image a specimen; light of shorter
wavelengths is capable of resolving greater detail than longer wavelengths.

There are three mathematical concepts which need to be taken into consideration when dealing
with resolution: ‘Abbe’s Diffraction Limit’, ‘Airy Discs’ and the ‘Rayleigh Criterion’. Each of
these are covered below in chronological order.

George Biddell Airy and ‘Airy Discs’ (1835)

George Biddell Airy (1801-1892) was an English mathematician and astronomer. By the 1826
(aged 25) he was appointed Professor of Mathematics at Trinity College and two years later, he
was appointed Professor of Astronomy at the new Cambridge Observatory. From 1835 to 1881he
was the ‘Astronomer Royal’ and he has a lunar and Martian crater named in his honor.

Also in the year 1835, he published a paper in the Transactions of the Cambridge Philosophical
Society entitled ‘On the Diffraction of an Object-Glass with Circular Aperture’. Airy wrote this
paper very much from the view of an astronomer and in it he describes “the form and brightness
of the rings or rays surrounding the image of a star as seen in a good telescope”. Despite writing
in a different scientific field, these observations are relevant to other optical systems and indeed,
the microscope

An Airy Disc is the optimally focused point of light which can be determined by a circular
aperture in a perfectly aligned system limited by diffraction. Viewed from above (Figure 1), this
appears as a bright point of light around which are concentric rings or ripples (more correctly
known as an Airy Pattern).

The diffraction pattern is determined by the wavelength of light and the size of the aperture
through which the light passes. The central point of the Airy Disc contains approximately 84% of
the luminous intensity with the remaining 16% in the diffraction pattern around this point. There
are of course many points of light in a specimen as viewed with a microscope, and it is more
appropriate to think in terms of numerous Airy Patterns as opposed to a single point of light as
described by the term ‘Airy Disc’.

The three-dimensional representation of the Airy Pattern as illustrated in the lower half of Figure
1 is also known as the ‘Point-Spread Function’.

Fig. 1: Typical phenomenom of an Airy Pattern, also known as Airy Disc, with its
central maximum point of light and the encircling diffractive rings.
Ernst Abbe and ‘Abbe’s Diffraction Limit’ (1873)

Ernst Abbe and ‘Abbe’s Diffraction Limit’ (1873)

Ernst Karl Abbe (1840-1905) was a German mathematician
and physicist and in 1866, he met Carl Zeiss and together they founded what was known as the
‘Zeiss Optical Works’, now known as Zeiss. In addition, he also co-founded Schott Glassworks
in 1884. Abbe was also the first person to define the term numerical aperture. In 1873, Abbe
published his theory and formula which explained the diffraction limits of the microscope. Abbe
recognized that specimen images are composed of a multitude of overlapping, multi-intensity,
diffraction-limited points (or Airy Discs).
In order to increase the resolution (d=λ/2 NA), the specimen must be viewed using either
shorter wavelength (λ) light or through an imaging medium with a relatively high refractive
index or with optical components which have a high NA (or, indeed, a combination of all of
these factors).

However, even taking all of these factors into consideration, the limits in a real microscope
system are still somewhat limited due to the complexity of the whole system, transmission
characteristics of glass at wavelengths below 400 nm and the achievement of a high NA in the
complete microscope. Lateral resolution in an ideal light microscope is limited to around 200
nm, whereas axial resolution is around 500 nm (for examples of resolution limits, please see
below).

John William Strutt and ‘The Rayleigh Criterion’ (1896)

John William Strutt, 3rd Baron Rayleigh (1842-1919) was an English physicist and a prolific
author. During his lifetime, he wrote an astonishing 466 publications including 430 scientific
papers. He wrote on a huge range of topics as diverse as bird flight, psychical research, acoustics
and in 1895, he discovered argon (for which he was later awarded the Nobel Prize in Physics in
1904).

Rayleigh built upon and expanded the work of George Airy and invented the theory of the
‘Rayleigh Criterion’ in 1896. The Rayleigh Criterion (Figure 2) defines the limit of resolution in
a diffraction-limited system, in other words, when two points of light are distinguishable or
resolved from each other.

Using the theory of Airy Discs, if the diffraction patterns from two single Airy Discs do not
overlap, then they are easily distinguishable, ‘well resolved’ and are said to meet the Rayleigh
Criterion (Figure 2, left). When the center of one Airy Disc is directly overlapped by the first
minimum of the diffraction pattern of another, they can be considered to be ‘just resolved’ and
still distinguishable as two separate points of light (Figure 2, mid). If the Airy Discs are closer
than this, then they do not meet the Rayleigh Criterion and are ‘not resolved’ as two distinct
points of light (or separate details within a specimen image; Figure 2, right).

Fig. 2: The limit of

resolution (defined by
the Rayleigh Criterion)
shown by the overlapping diffraction patterns of two single Airy Disks: Left: Well resolved Mid: Just resolved Right: Not resolved

How to calculate the resolution of a microscope

Taking all of the above theories into consideration, it is clear that there are a number of factors to
consider when calculating the theoretical limits of resolution. Resolution is also dependent on the
nature of the sample. Let’s look at calculating resolution using Abbe’s diffraction limit and also
using the Rayleigh Criterion.

Firstly, it should be remembered that:

NA= n x sin α

Where n is the refractive index of the imaging medium and α is half of the angular aperture of
the objective. The maximum angular aperture of an objective is around 144º. The sine of half of
this angle is 0.95. If using an immersion objective with oil which has a refractive index of 1.52,
the maximum NA of the objective will be 1.45. If using a ‘dry’ (non-immersion) objective the
maximum NA of the objective will be 0.95 (as air has a refractive index of 1.0).

Abbe’s diffraction formula for lateral (i.e. XY) resolution is:

d= λ/2 NA
Where λ is the wavelength of light used to image a specimen. If using a green light of 514 nm
and an oil immersion objective with an NA of 1.45, then the (theoretical) limit of resolution will
be 177 nm.

Abbe’s diffraction formula for axial (i.e. Z) resolution is:

d= 2 λ/NA2

Again, if we assume a wavelength of 514 nm to observe a specimen with an objective of NA

value of 1.45, then the axial resolution will be 488 nm.

The Rayleigh Criterion is a slightly refined formula based on Abbe’s diffraction limits:

R= 1.22 λ/NAobj+NAcond

Where λ is the wavelength of light used to image a specimen. NAobj is the NA of the objective.
NAcond is the NA of the condenser. The figure of ‘1.22’ is a constant. This is derived from
Rayleigh’s work on Bessel Functions. These are used for calculating problems in systems such
as wave propagation.

Taking the NA of the condenser into consideration, air (with a refractive index of 1.0) is
generally the imaging medium between the condenser and the slide. Assuming the condenser has
an angular aperture of 144º then the NAcond value will equal 0.95.

If using a green light of 514 nm, an oil immersion objective with an NA of 1.45, condenser with
an NA of 0.95, then the (theoretical) limit of resolution will be 261 nm.

As stated above, the shorter the wavelength of light used to image a specimen, then the more
detail will be resolved. So, if using the shortest visible wavelength of light of 400 nm, with an oil
immersion objective with an NA of 1.45 and a condenser with an NA of 0.95, then R would
equal 203 nm.

To achieve the maximum (theoretical) resolution in a microscope system, each of the optical
components should be of the highest NA available (taking into consideration the angular
aperture). In addition, using a shorter wavelength of light to view the specimen will increase the
resolution. Finally, the whole microscope system should be correctly aligned.

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.

The angle m is one-half the angular aperture (A) and is related to the numerical
aperture through the following equation:

Numerical Aperture (NA) = n (sin m)

where n is the 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. Many authors substitute the
variable a for m in the numerical aperture equation. From this equation it is obvious that when
the imaging medium is air (with a refractive index, n = 1.0), then the numerical aperture is
dependent only upon the angle m whose maximum value is 90°. The sin of the angle m,
therefore, has a maximum value of 1.0 (sin (90°) = 1), which is the theoretical maximum
numerical aperture of a lens operating with air as the imaging medium (using "dry" microscope
objectives).

Objective Lenses

In microscopy, the objective lenses are the optical elements closest to the specimen. The
objective lens gathers light from the specimen, which is focused to produce the real image that is
seen on the ocular lens. Objective lenses are the most complex part of the microscope due to
their multi-element design. It is this complexity that makes the objectives the most important
components of the device.

Objective lenses are responsible for:

 Primary image formation
 Determine the quality of the image produced
 The total magnification
 The overall resolution

The refractive objectives are the most common objectives. With refractive objectives, light is
bent (refracted) by the optical elements, which are designed in a manner that reduces back
reflections thereby improving the overall light passing through. These types of objectives are
often used in applications that require resolution of highly fine details. For refractive objectives,
designs may range from two elements in the basic achromatic objectives to fifteen elements in
plan-apochromatic objectives.

Meaning of Micrometry:
Micrometry is the science in which we have some measurement of the dimensions of an object
being observed under the microscope. The method employs some special types of measuring
devices which are so oriented that these can well be attached to or put into the microscope and
observed. The object, to be measured, is calibrated against these scales.

Once we are observing an object under a microscope by the 5X objective and the 10X eyepiece
we say that the image that we are able to perceive is 5 × 10 = 50 times of the object.

We get the magnified view no doubt and also that it is the perfect coordination of the
dimensions, but to find out the exact size of the object will need precision and that is achieved
through the application of some small scales called micrometers.

Types of Micrometry:

1. Stage Micrometer:
As is clear from its name it is for the measurement on the stage of the microscope where an
object is to be kept. This micrometer is of a slide’s shape and size and has amount of very finely
graduated scale. The scale measures only 1 mm and has a least count of 0.01 mm, i.e. 1 mm
region is divided into 100 divisions. As 1 mm has 1000µ, one division of stage micrometer is
equivalent to 10µ.

2. Ocular Meter:
This micrometer is used inside the eyepiece. The upper eye lens is unscrewed and the ocular
meter is put into the tube of eyepiece, and the eye lens is again replaced in its original position.
There are usually 50 or 100 divisions in the ocular meter which are engraved on the glass.

V. EVALUATION

MABALACAT CITY COLLEGE

INSTITUTE OF ARTS, SCIENCES AND TEACHER
EDUCATION
Mabalacat City, Pampanga

Module 1 – BSSE 119 - BIOTECHNIQUES

The Microscope
Compiled and Prepared by Rowena V. Aldana 1st Semester A.Y. 2020-2021
Name: _______________________________ Score:____________________
Course/Section:____________________________ Date: ____________________

ACTIVITY 1: Encircle the letter of the correct answer.

1. The parts labelled Y and Z in the diagram are
a. the eyepiece and mirror respectively
b. the objective lens and the turret respectively
c. the mirror and the eyepiece respectively
d. the eyepiece and the objective lens respectively
2. Look at the image of a microscope in the diagram. Identify which part A, B or C
is the stage, and select the source of light which illuminates the object being
viewed with this microscope.
a. C is the stage and the object is illuminated by sunlight.
b. C is the stage and the object is illuminated by a light bulb.
c.  A is the stage and the object is illuminated by a light bulb.
d. B is the stage, and the object is illuminated by sunlight.
3. This is turned to bring the object being viewed closer.

a. Focus knob
b. Objective lens
c. Eyepiece lens
d. Coverslip
4. The diagram shows a laboratory microscope.
What are the functions of parts labelled A and B?
a.  A: To magnify the object.
B: To illuminate the object being viewed.
b. A: To magnify the object.
B: To hold the slide on the stage.
c. A: To adjust the fine focus.
B: To hold the slide on the stage.
d. A: To adjust the coarse focus.
B: To illuminate the object being viewed.
5. If the eyepiece of a microscope magnifies by 10, and the objective lens magnifies by 4, then what
is the total magnification of the microscope?
a. 2.5 b. 0.4 c. 40 d. 14
6. From the following list, choose the correct order in which light passes through it.
a. Mirror ➞ objective lens ➞ eyepiece ➞ slide
b.  Slide ➞ eyepiece ➞ objective lens ➞ mirror
c. Mirror ➞ slide ➞ objective lens ➞ eyepiece
d. Mirror ➞ eyepiece ➞ objective lens ➞ slide
7. The diagram shows a microscope. What is the part labelled X and what is its function?
a. The coarse adjustment and its function is to allow you to see a clear, magnified image.
b. The mirror and its function is to shine light on the glass slide.
c. The objective lens and its function is to allow you to see a clear, magnified image
d.  The stage and its function is to hold the glass slide.
8. To focus on a specimen, it is best to start with which objective lens?
a. Lowest magnification
b. Highest magnification
c. Intermediate magnification
 d. None of the above
9. Which part of a microscope do you look through to see an object magnified? 
a. Objective lens
b.  Focus knob
c. Eyepiece lens
d. Stage
10. When viewing a sample of onion cells using a microscope, the student placed the cells on a slide
and placed a coverslip over it. What is the purpose of the coverslip?
a.  To protect the onion.
b. To keep the cells flat.
c. So the onion cells do not fall of the slide.
d. To get a better view of the cells

MABALACAT CITY COLLEGE

INSTITUTE OF ARTS, SCIENCES AND TEACHER
EDUCATION
Mabalacat City, Pampanga

Module 1 – BSSE 119 - BIOTECHNIQUES

The Microscope
Compiled and Prepared by Rowena V. Aldana 1st Semester A.Y. 2020-2021

Name: _______________________________ Score:____________________

Course/Section:____________________________ Date: ____________________

ACTIVITY 2: Encircle the letter of the correct answer.

1. Why do oil immersion objectives have higher numerical apertures than dry (non-oil) lenses?

a. They are used closer to the specimen; therefore, their lenses intercept light at angles dray
lenses would miss.
b. The oil used with the lenses prevents light from being refracted away from the objectives
as does occur with the dry lenses.
c. The oil used with the lenses increases the difference between the refractive indices of the
slide and the medium between the slide and the objective lens.
d. A and B
 2. Which microscope lens does not magnify the imaged formed?
a. magnetic lens c. objective lens
b. ocular lens d. condenser lens
3. If the minimum image size the human eye can detect is 0.1 mm, what is the minimum
magnification needed to make an object measuring 1 um visible?
a. 10x c. 1,000x
b. 100x d. 10,000x
4. If the total magnification of a microscope is 2000X with the use of a 10X ocular lens, what is the
magnification of the objective lens?
a. 10X c. 200X
b. 20X d. 2000X
5. What is the total magnification of a 10x ocular lens and a 45x objective lens?
a. 15x c. 150x
b. 45x d. 450x

B. Answer the following

1. State 2 procedures which should be used to properly handle a light microscope.

2. Explain why the light microscope is also called the compound microscope.
VI. REFERENCES:
https://www.biologydiscussion.com/micrometry/micrometry-meaning-and-types-with-diagram-
biology/56994\
https://www.microscopemaster.com/parts-of-a-compound-microscope.html
https://www.youtube.com/watch?v=RKA8_mif6-E&t=65s
https://www.youtube.com/watch?v=6jkK5fhZesA&t=83s
https://www.youtube.com/watch?v=pFSvM8KD5BY&t=95s
https://www.youtube.com/watch?v=1mK9EmY_r1o&t=18s
https://www.youtube.com/watch?v=erMdBKj9E3I

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