Cell Biology

October 1, 2017 , 2017

Microscope: Our windows into the Cell

Zarlene A. Sierra
Science Department, College of Natural Sciences and Mathematics, Mindanao State University
General Santos City 9500

Abstract
Keywords: Brine Shrimp Lethality Assay, Catharanthus roseus, LC50 Potent, Cytotoxicity,
Allium cepa Assay, Genotoxicity

1. Introduction

All living organisms are composed of one or more cells. It is the basic morphological and
functional unit of all living things. It has the capacity to perform all functions and is therefore
capable, under a favorable environmental conditions, of independent existence. And in fact,
many of them, are bacteria which can be either beneficial or harmful, and it can also be a
parasites. But the problem is, cells are small that they are either barely visible or not visible
with the naked eye alone. So how can we be able to identify and classify organisms? How can
we be able to know and learn about us? -- Thats where microscopy takes place.

Before the 1600, no one knew that large organisms are made up of many small, living units
that we call cells. Cells are typically between 1 and 300 micrometers (m) in diameter. A
micrometer is one millionth of a meter. To put that size into perspective, a diameter of 300 m
is only about one third of a millimeter, or about one hundredth of an inch. But before we will
dig deeper into the different types of microscope we have nowadays, let us first go back to
those persons behind this technology the pioneers of Microscopy. Robert Hooke was a
scientific virtuoso, making discoveries in such diverse fields as biology, physics, and
astronomy. According to a biographer in 1705, Hooke was an active, restless, indefatigable
geniusand always slept little to his death, seldom going to sleep till two, three, or four oclock
in the morning. Hooke maintained an association with a Dutch amateur scientist named
Antoine Van Leeuwenhoek (1632-1723). Van Leeuwenhoek made hundreds of tiny, single-
lens microscopes only a few inches high. To us they look more like miniature violins than
microscopes, but they provided magnifications of up to 500 times with minimal distortion. This
differed dramatically from instruments commonly used at the time, which were tubes up to
three feet tall. Such tubes contained two or more lenses usually made by blowing glass into
various shapes, which were then intentionally dropped on the floor in the hope of obtaining
fragments with lens-like optical properties. These lenses did indeed magnify, but they also
distorted, and the addition of a second lens simply magnified the distortion. In contrast, Van
Leeuwenhoek became an important figure in microscopy, the use of microscope, because he
was the first person to make single-lens microscopes with high magnifications that did not
distort what was being observed. Van Leeuwenhoek found microbes virtually everywhere he
looked. There are, he said, more animals living in the scum on the teeth in a mans mouth
than there are men in a whole kingdom. In November 1677, Hooke confirmed Van
Leeuwenhoeks starting claim that many tiny animals lived in drops of pond water. Van
Leeuwenhoek was not surprised that many people doubted his observations. I cant wonder at
it, he wrote, since tis difficult to comprehend such things without getting a sight of em. In
his confirmation, Hooke wrote that the animals were, perfectly shaped with such curious
organs of motion as to be able to move nimbly, to turn, stay, accelerate, and retard their progress
at pleasure. Van Leeuwenhoeks and Hookes contributions to microscopy opened up a new
frontier for science.

Microscopes, of course, serve as our windows into the cell. The magnifying power of light
microscope has improved considerably over the years, but the basic principle remains the same.
A light microscope uses glass lenses to bend the path of visible light, thereby producing a
magnified image. A modern light microscope can resolve objects 200 nanometers (nm) apart,
or 1,000 times better than the human eye. The development in 1939 of the electron microscope,
which focuses electrons with magnetic lenses, made possible a wealth of discoveries aout cells.
The transmission electron microscope (TEM) reveals structures within cells by passing elctrons
completely through a thin section of tissue. A TEM can magnify objects up to about 100,000
times, enabling scientists to view cell structures as small as 2 nm, a great advance over the light
microscope. A second type of electron microscope, the scanning electron microscope (SEM),
bounces electrons off a specimen to reveal the surface structure, often a detailed 3-D view. A
SEM can magnify up to about 200,000 times, giving detailed views of cells, group of cells, and
small organisms or parts of organisms. Overall, light microscopes and electron microscopes
are most useful for structures that are just beyond the human eye.

But, we will focus more on light microscope, because the light microscope (compound light
microscope), which uses light as a source of illumination, will be employed in this lab. In this
activity, and through this microscope, we will be able to determine the magnifying power
(Total magnification) when using different objective lenses, the orientation of letter e when
viewed in scanner, LPO, and HPO, and draw different morphological bacterial cells (Amoeba
proteus and Enterobacter aerogenes), cheek cell, Onion cell, and Elodea cell. This are just the
basic laboratory activity for us to learn the basic about microscopes and cells. But it is as
important as viewing more complex smears using more complex microscopes because it will
give us a background about microscopes and cells.

2. Method and Materials

In this activity, we will be needing: first, of course, the microscope specifically the Leica DM
750 Compound Microscope. Second is the prepared written letter e in a very small piece of
paper that can be fit in slide covered by coverslip. Prepared bacterial smears, A cheek cell, an
onion (onion cell), for determining the visible components of plant cell, and Elodea cell to view
plant cell specifically the mitochondria and vacuole.

2.1 Determining the Magnifying Power

In determining the magnifying power, we first know the different magnification of the ocular
lens, and objectives (Scanner, LPO, HPO, and OIO). Next is we just calculated the Total
magnification by using this formula:

Total Magnification = Ocular Magnification X Objective lens

2.2 Image Orientation and focusing

Here, we used the prepared written letter e in a piece of paper, put it in a slide and covered it
with the coverslip, and wet it by water. Observed it in Scanner, low power and high power
objective. Also, we observed what happened when we moved the slide to the left and right
using slide movement knobs. Next, we observed what happened to the light intensity when e
switched it from low to high power. And lastly, we observe what happened to the working
distance when we increased the magnification.

2.3 Diameter of the Field of View

In this part of the activity, we measured the field of view at scanner power and low power using
a ruler and calculated the high power diameter using this formula:

Diameter of the field of view X Scanning power total magnification

Total magnification

Remember: The total magnification will depend on what objectives diameter you want to get.

2.4 Depth of Field

Here, we observed three colored threads and observed if it is focus in low power and if it still
focus when switched to high power.

2.5 Prokaryotic and Eukaryotic cells

In this part, we viewed bacterial smears (Amoeba proteus and Enterobacter aerogenes) in Oil
Immersion, drew and labeled their different parts. Next, we viewed cheek cell which from one
my classmate (scraped from cheeks) stained with Methylene Blue in high power objective and
then drew and labeled the visible components (cell membrane, cytoplasm, nucleus, nuclear
envelope, mitochondria and nucleus). Next one is we viewed onion cell and find cell wall,
cytoplasm, nucleus, nuclear envelope, mitochondria and nucleolus if visible, then drew and
labeled it under HPO. And lastly, we viewed Elodea cell, draw and labeled components if
visible in HPO, especially the cell wall, cytoplasm, chloroplast, and vacuoles.

a.

3. Results and Discussion

This part of the paper talks about the results on what we did in chapter 2. This includes the:
Determining the Magnifying Power, Image Orientation and focusing, Diameter of the Field of
View, Depth of Field, Prokaryotic and Eukaryotic cells. It also includes further discussion
about the above mention, and answer to those follow up and guide questions.
3.1 Determining the Magnifying Power
In getting the magnification, the formula below will be used. Magnification of ocular lens and
objective lens are also provided.
Formula:
Total Magnification = Ocular magnification X Objective magnification
Ocular lens: 10x; Scanning (4x); LPO (10x); HPO (40x); OIO (100x)

1a. Scanning Power: 10x (4x) = 40x

1b. Low Power: 10x (10x) = 100x
1c. High Power (Dry): 10x (40x) = 400x
1d. Oil Immersion: 10x (100x) = 1000x

So, Scanning power will magnify the organism 40x, Low power objectives (LPO) will magnify
organisms 100x, High power objectives will magnify organisms 400x, and Oil Immersion
objective (OIO) will magnify organisms 1000x when viewed in a microscope.

3.2 Image Orientation and Focusing

Fig. 3.2.1. Letter e; Mag: 40x

Fig. 3.2.1 Letter e. This figure shows letter e on scanning power with a magnification of
40x. Under the microscope, it appears to be inverted or opposite on how it is set-up on the slide.

So what direction in the field of view did the letter e move when you moved the slide
slightly to the left or right using the slide movement knobs? Up or down? When you moved
the slide to the left, the direction of the image moves to the right. When you move the slide to
the right, the direction of the image will move to the left. On the other, when you move the
slide downwards, the image will move towards you, but if you move it upward, the image will
move away from you.

Fig. 3.2.1. Fig. 3.2.1. Fig. 3.2.1.

Letter e; Letter e; Letter e;
Mag: 40x Mag: 40x Mag: 40x
(a) Letter e; Mag: 40x (b) Letter e; Mag: 100x (c) Letter e; Mag:
400x

Figure 3.2.2. The view Letter e under three different magnification. (a) View of Letter
e under scanning power magnified 40x. On scanner objective, the whole inverted letter e
can be seen, and the field of view is wider. (b) View of Letter e under low power objective
(LPO) magnified 100x. Under LPO, we can see in the figure, the whole part of letter e cant
be seen, some part of it, especially on the tail part are removed, which means we cannot see it
on the microscope. (c) View of Letter e under high power objective (HPO) magnified 400x.
On HPO, you cant now identify a letter e, just a colored black like tube.

But what will happen to the light intensity when you switched from low to high power?
What can you do to adjust for this change? What happened to the working distance
(distance between the slide and the objective lens) when you increased magnification?
When we switched from low power to high power, it means the magnification also increases,
and it means that the light intensity decreases. And in order to adjust from this change, we need
to raise the light intensity by turning the light control knob clockwise or to the right because as
the magnification increases, the light intensity decreases. And as the magnification increases,
the working distance increases.

3.3 Diameter of the Field of View

Determining the field of view is important in order for us to determine the size of our specimen.
When we observe the specimen, we must estimate its size by comparing it with the size of the
field.

Size of Field of view X Scanning power total magnification

Total magnification

Remember: The total magnification will depend on what objectives diameter you want to get.

Scanning power diameter: 4.4 m

Low power diameter: 1.76 m
High Power diameter: 440 m

Equation:
Estimated size of field of view: 4.4 mm

High power diameter: Size of Field of view X Scanning power total magnification
Total magnification
= 4.4 mm X 40x = 0.44 mm
400x
After you have solved the diameter, convert it m.
Note: 1mm = 1000 m
0.44 mm x 1000 m = 440 m

It is really important to know the diameter of the field of view, because we can use it to
determine or estimate the size of the organism you are viewing at that given magnification.

3. 4 Depth of Field
We view three colored threads in low power and we tried if it is focused. And yes, it were all
focus. And it you view in on the microscope, from top to bottom, it is the arrangement: Yellow,
red, and blue. And if we are viewing organisms under LPO, we need to make sure that it is
focus before switching it to high power because microscopes are parfocal, once you have
focused carefully with the low power objective, the other objective will be nearly in perfect
focus.

B. Prokaryotic and Eukaryotic cells

Prokaryotic and Eukaryotic cells differ in so many ways, from the size or structure of the cell,
their genetic material, their DNA synthesis or replication, Transcription, and cell division.
Prokaryotic,

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Samuel H. Cohen, a member of the Microscope Society of America and a fellow of the Royal
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