UNITY AND

DIVERSITY

UNIT

AREA OF STUDY 1

Cells in action
Chapter 1 Cells: discovery and exploration
Chapter 2 Structure and function of cells
Chapter 3 Composition of cells
Chapter 4 Cell replication

Cells: discovery and

exploration

HIP

Image

HIP

KEY KNOWLEDGE
This chapter is designed to enable students to:
appreciate the historical development of microscopy techniques
investigate current and emerging technologies in light and electron microscopy

SyCoP

understand the importance of technological advances to our knowledge of life

forms and cells.

Figure 1.1 Examination of high-resolution three-

dimensional brilliant fluorescence images is now possible

with current stereomicroscopes such as this SteREO
Lumar.V12 manufactured by Carl Zeiss Pty Ltd. The
stereomicroscope has lenses specially developed for use with
fluorescence and its operation is completely motorised. The
focus of an object can be rapidly set and precisely reproduced
2 NATURE OF BIOLOGY BOOK 1
with the use of human interface panels (HIP). A system control

panel (SyCoP) is designed for use by either a right- or

left-handed person and combines joystick, buttons and a
touch screen a design similar to a computer mouse so
that the operator can control the microscope while still
viewing through the eyepiece. In this chapter, we will
discuss significant historical developments in microscopy
techniques and the latest advancements in microscope
technologies.

Life on Earth and beyond?

Is there (or was there ever) life on Mars?
At the turn of the twentieth century, an American astronomer, Percival Lowell
(18551916), drew maps of the surface of planet Mars that showed intricate
patterns of linear structures that he called canals. He argued that these canals
were not natural features but were artificial constructions produced by intelligent life. Figure 1.2a
shows Lowells drawings of canals on Mars. Figure
1.2b shows a typical area on the surface of Mars as
revealed by the Viking Lander in 1976. Definitely
no canals! Definitely no evidence of life, intelligent
or otherwise!

(a)

Figure 1.2 (a) Canals on Mars based on observations made from

Earth by Lowell in the early 1900s, and (b) the surface of Mars as
revealed by the Viking Lander in 1976. Can you suggest a possible
reason for Lowells observations being flawed?
(b)

ODD FACT
In July 1976,
when the Viking Lander reached
the surface of Mars, it became
the first spacecraft to land on
the surface of another planet.

Figure 1.3 Scanning electron

micrograph image of part of a meteorite
(known as ALH84001) from the surface
of Mars that landed in the Antarctic.
While the elongated structures look like
microbes (tiny living organisms) they
are not universally accepted as being
fossilised microbes.

The Viking Lander carried instruments to test for the existence of living organisms on Mars (note the trenches dug by the soil retrieval scoop in figure 1.2b),
but the results of the tests were inconclusive.
Then, in 1996, sensational headlines worldwide publicised the claim by NASA
scientists that life once existed on Mars. This claim was based on studies of a
meteorite that originated from that planet. The evidence included the presence of
tiny structures within the meteorite (see figure 1.3) that were said to be fossilised
microbes (tiny living organisms). However, other scientists disputed this claim
and argued that these microbe-like structures could be produced by chemical
reactions. Again, the evidence for life on Mars was inconclusive.
Another development occurred in January 2004
when two Rovers landed on the surface of Mars to
study its rocks and minerals. Data from these Rovers
provided evidence that liquid water once existed on
Mars. We know that liquid water is essential for life.
We also know that microbes can survive in extreme
environments on Earth, such as in rocks deep below
ground, in ice-sealed lakes, in glaciers high on mountains and in cold dry valleys of Antarctica. Based on
these facts, it remains possible that life does or did
exist on Mars.
There are plans to launch a Mars Science Laboratory from Earth to Mars in December 2009. This
mobile laboratory will search for evidence of life past or present on Mars
using instruments that can detect organic compounds, such as proteins and amino
acids, that are made only by living organisms.
Scientists expect that, if life exists now or existed in the past on Mars, this
extraterrestrial life will be like the microbes that live today in extreme environments on Earth. Microbes, like all living things, are organised into microscopic
compartments known as cells. Each microbe typically consists of just one cell
and the internal contents of each cell are separated from the external environment
CELLS: DISCOVERY AND EXPLORATION

by a membrane boundary. The strongest direct evidence for past or present extraterrestrial microbial life on Mars would be the discovery of structures that can
without any doubt be identified as cells.
Let us now look in more detail at the historical development of ideas and technological advances that have contributed to our knowledge and understanding of
life forms, and their living compartments or cells.

Cells and microscopes:

an introduction
Cells are the basic structural and functional units of all living things (figure 1.4).
Although most cells are too small to be seen with the unaided eye, microscopes
give enlarged images of cells and the structures they contain, and make it possible
for us to examine cells with great detail.

Figure 1.4 Most human cells

typically range in diameter from about
8 to 25 micrometres (Mm) or 0.008 to
0.025 mm. In comparison, a hair from
a mans beard is about 200 Mm
(0.2 mm) wide. Typical bacterial cells
range from 0.11.5 Mm and giant
amoeba are about 1000 Mm wide.
Note the sizes of various kinds of cells
(1 mm = 1000 Mm). Cells are not drawn
to scale.

Cell type

Example

Size

animal

frog egg

1500 Mm

human egg (note the

relative size of sperm)

200 Mm

human white blood cell

25 Mm

human red blood cell

8 Mm

fungus

yeast cell

5 Mm

bacteria

Staphylococcus
(causes infections
such as boils)

1 Mm

Diplococcus pneumoniae
(causes pneumonia)

0.1 Mm

Treponema pallidum
(spiral causes
syphilis)

0.3 Mm wide
and
10 Mm long

epidermal leaf
cell

200400 Mm

plant

The development of microscopes over the centuries has depended on the

development of glass, then on glass being made into lenses, the development of
different kinds of lenses and their assembly to form microscopes.
4

NATURE OF BIOLOGY BOOK 1

Table 1.1 Some important

milestones in the development of
microscopes

The increase in our understanding of cells has paralleled:

the improvements in and development of new kinds of microscopes
the variety of different techniques available, including stains, sectioning and
using different kinds of light.
The advanced microscopes of today have a history dating back more than
three thousand years to when the first glass was made by Phoenician sailors. A
summary of some of the important steps in the development of the microscope
and our understanding of cells is presented in table 1.1.

Date or period

Person and development

> 3000 years ago

Glass beads first made by Phoenician sailors, who were from an area now known as Lebanon

250 BCAD 100

In China, the first recorded uses of optical lenses

AD 79
(excavated 1748)

People of Pompeii used glass-crystal lenses

12001250

Robert Grosseteste, Bishop of Lincoln, UK, made a primitive but functional magnifying glass.

1590s

Dutch lens-makers, Hans Janssen and his son Zacharias, used two lenses to develop the first compound
microscope, called telescope by some writers.

16051619

Cornelius Drebbel (15721633), a Dutch/English inventor of many scientific instruments, developed a

machine for grinding lenses and improved the quality of compound microscopes.

16051614

Galileo Galilei (15641642), an Italian, refined the Janssen microscope into a high-quality astronomical
telescope. He also further developed the microscope and may have been the first to examine and describe
living tissue. He described the cuticle of a fly as being covered in fur.

between 1605
1610

Galileo was a prominent member of the Accademia dei Lincei (Academy of the Lynx) that introduced the
term microscopio a lens for the examination of very small objects.

1665

Englishman Robert Hooke (16351703) published Micrographia. He describes cells in a piece of cork
(page 6 and figure 1.5) and draws many cell types. Hookes microscope could magnify 1442 times.

1674

Antony van Leeuwenhoek (16321723), a Dutch cloth merchant, built a microscope with a magnifying
range from 50 to 300 times. He was the first to make descriptive drawings of protozoa, bacteria,
spermatozoa and red blood cells (page 6 and figure 1.6, page 7).

1733

Englishman Chester Moor Hall used lenses made of different kinds of glass to invent the achromatic lens
that removed many of the optical distortions of previous lenses.

1738

German Johann Lieberkuhn added a metal reflector to the microscope to increase light falling on a
specimen.

1831

Robert Brown (17731858), a Scottish botanist and naturalist, described the nucleus in orchid cells (figure
1.7, page 7 and page 8).

1838

Two Germans, botanist Matthias Schleiden (18041881) and zoologist Theodor Schwann (18101882),
suggested that cells are the basic structural units of all plant and animal matter.

1851

Binocular microscope (viewing with two eyes) constructed by Professor Riddell

1878

Germans Ernst Abby and Carl Zeiss produced improved oil-immersion microscope lenses that significantly
increased the ability to magnify cells (figure 1.10, page 11).

19311933

German Ernst Ruska developed the electron lens and used several to make the first electron microscope.

1936

Swedish Torbjorn Oskar Caspersson used an ultraviolet microscope to study cells.

1938

Dutch Fritz Zernike built the first phase contrast microscope enabling examination of transparent cells and
micro-organisms without the need to stain or kill.

1955

Marvin Minsky of the USA invented the confocal scanning microscope.

1969

Scientists in Holland, Britain and America developed confocal laser scanning microscopy. Americans
Paul Davidovits and David Egger announced they were able to optically section thin slices of threedimensional specimens such as a cell (as in figure 1.15, page 12).

2003

PlasDIC is a special form of a differential interference contrast microscope in which special prisms are
used to reveal high-resolution, three-dimensional details of a specimen illuminated with non-polarised light
(figures 1.21 and 1.22 on page 16).

2004

Laser scanning microscopy LSM 5 LIVE scans living cells at speeds of up to 1010 frames per second.
Allows a better understanding of cellular processes and study into cellular interaction mechanisms.

CELLS: DISCOVERY AND EXPLORATION

In this chapter, we will consider some of the people and technologies, and their
contribution to our understanding of cells. We will also consider the characteristics of the following tools used for viewing cells.
Light microscopes:
Simple light microscope
Compound light microscope
Phase-contrast microscope
Fluorescence microscope
Scanning confocal microscope
PlasDIC microscope
Electron microscopes:
Transmission electron microscope
Scanning electron microscope

ODD FACT
Robert Hooke was
a scientist, inventor and
architect who drew up plans for
the rebuilding of London after
the Great Fire of 1666. Hooke
built the vacuum pump that
Boyle used in his experiments
on the pressure and volume of
gases. Hooke also formulated
the law that describes the
behaviour of springs when
stretched.

Cells: an historical overview

In his book, Micrographia, published in 1665, English scientist Robert Hooke
(16351703) describes how he used a microscope to examine thin slices of cork
from a tree and saw small box-like compartments that he called cells. Robert
Hooke is credited as the person who discovered cells. In fact, Hooke was not
looking at living cells. What he saw were the remains
of dead and empty plant cells (see figure 1.5). However,
Hookes observations were important because he was
the first to realise that this plant material had an organised structure at the microscopic level.

Figure 1.5 (a) The microscope

that Robert Hooke built and used to
examine thin slices of plant material.
What name did he give to the minute
building blocks that he saw? What
light source might Hooke have used
for this microscope? The specimen for
examination was placed on a specimen
holder. (b) First drawings made in
1665 of cells from a thin piece of
cork. Were these living or dead cells?

In 1674, a few years after Hooke discovered cells, a Dutch cloth merchant,
Anton van Leeuwenhoek (16321723), used a simple microscope (see figure
1.6a, page 7) to observe material that he scraped from between his teeth. After
examining this material, he wrote:
in the said matter there were very many little living animacules, very prettily
a-moving.

What Leeuwenhoek saw were probably the first bacterial cells to be viewed (see
figure 1.6b). Although Leeuwenhoeks original interest with microscopes was
to examine fibres in the cloth he traded, he was inspired by the publication of
Hookes Micrographia.
6

NATURE OF BIOLOGY BOOK 1

(a)

(b)

(c)

Figure 1.6 (a) The simple microscope built by Leeuwenhoek.

The specimen was placed on the tip of a pin that acted as a
specimen holder. The lens, only two millimetres wide, was ground
out of a quartz crystal and was fitted into a hole in a metal plate.
The instrument was held up to the eye and the specimen viewed
through the lens. (b) Some of the little animacules seen by
Leeuwenhoek. These were various bacteria. (c) Algal and other
cells from pond water as drawn by Leeuwenhoek
(a)

(b)

Figure 1.7 (a) Wax medallion

of Robert Brown, made in 1852
(b) One of the microscopes used by
Brown in his observations of pollen
and other plant cells. Note the
mirror (closest to the base) and the
fine-adjustment knob for movement
of the specimen platform above it.
CELLS: DISCOVERY AND EXPLORATION

ODD FACT
Robert Brown was
the botanist who accompanied
Sir Joseph Banks on the
Investigator, when Captain
Matthew Flinders charted the
southern coast of Australia from
1801 to 1805. Brown and Banks
collected nearly 4000 specimens
of different species of plants,
most of them unknown to Western
science at the time. Brown also
discovered molecular movement,
now called Brownian movement.

Leeuwenhoek built over 50 simple microscopes to examine material from different sources. Figure 1.6c shows Leeuwenhoeks drawings of some of the algal
and other cells he observed in pond water.
More than 150 years later, in 1831, Robert Brown (17731858), a Scottish
botanist (see figure 1.7, page 7), was involved in a dispute about how pollination
and fertilisation occurred in plants. During his studies with orchids, on 13 June
1831 he made a note that:
It appears that each cell has on its inner side a spherule or at least orbicular
corpuscle

Brown called this structure the nucleus of a cell. Others, including Leeuwenhoek,
had observed nuclei but Brown was the first to introduce the concept of a nucleated cell as the unit of structure in plants. Brown had no idea about the importance
of the nucleus and had some doubt about whether each cell needed one.

Recognising the pattern: the Cell Theory

ODD FACT
Schleiden was initially
educated as a barrister. Because
of his lack of success in this
profession, he attempted
suicide, shooting himself in
the forehead, but recovered.
Schleiden then turned to
the study of natural science
and medicine and became a
professor of botany.

By the early 1800s, the accepted idea was that plants and animals were composed
of globules, called cells, and formless material. Brown had enhanced this idea by
describing nuclei in cells of orchid plants. These views were to be extended by
two German biologists.
In 1838, a German botanist, Matthias Schleiden (18041881), suggested that
cells were the basic structural unit of all plant matter. A German zoologist, Theodor
Schwann (18101882), independently proposed that animals were aggregates of
cells arranged according to a definite law. In sharing their ideas over dinner in
October 1838, the two biologists came to recognise that both plant and animal
tissues have a cellular organisation. Nearly 200 years after Hooke first described
cells, the basic structural pattern of living things was finally recognised.
The recognition that all kinds of living things share a common structural unit
the cell provided the foundation of one of the major unifying themes of
biology. All living things are composed of cells and substances produced by cells
or developed out of cells. Because of this unity of structure, results of studies of
cells from one type of organism can be used to make predictions about cells from
other kinds of organisms. Schwann wrote in 1839:
The elementary parts of all tissues are formed of cells in an analogous,
though very diversified manner, so that it may be asserted, that there is one
universal principle of development for the elementary parts of organisms,
however different, and that this principle is the formation of cells.

This basic idea arising from the work of Schwann and Schleiden, published in 1839, is known as the Cell Theory:
All living things consist of one or more organised structures that are called
cells or of products of cells.
Cells are the basic functional unit of life.

Figure 1.8 According to one theory,

living things could arise from dead
matter; for example, leaves could
become birds or fish, depending on
where they fell.

NATURE OF BIOLOGY BOOK 1

A German doctor, Rudolf Virchow (18211902) added to the understanding of cells by providing a new answer to the question: How are new
living things produced? Past answers to this question included spontaneous generation, the idea that living things could arise from nonliving matter or dead matter. Another idea was that living things developed
from globules that gathered to form a compact mass and then became
organised into cells.
In 1858, Virchow challenged these old ideas with his concept of biogenesis
(from bio = life; genesis = origin, creation). He proposed that new cells come
from existing cells, and in one of his famous lectures said:

 no development of any kind begins de novo [from new] Where a cell arises,
there a cell must have previously existed just as an animal can spring only from
an animal, a plant only from a plant No developed tissue can be traced either
to any large or small simple element, unless it be a cell.

Virchows contribution extended the Cell Theory to include the basic concept:
New cells are produced from existing cells.

In 1862, the French biologist, Louis Pasteur (18221895) carried out experiments that conclusively disproved the old idea of spontaneous generation, and
supported the view that new cells are produced by existing cells.

Life span of cells

The terms unicellular and
multicellular refer to organisms
built of one or more building
blocks respectively.

ODD FACT
Spray-on skin
cells are now used in the
treatment of some burns
(see chapter 4, page 77).

Cells of a multicellular organism do not necessarily live as long as the organism

itself. Some cells have a relatively short life and are constantly being replaced.
The average life spans of some human cells are as follows:
stomach cells 2 days
mature sperm cells
23 days
skin cells
2035 days
red blood cells
about 120 days.
A person can make a blood donation because the cells removed can be replaced
by new cells. Skin can be taken from one part of a persons body and grafted onto
another area where the skin tissue has been completely destroyed. Skin cells
from the undamaged area will reproduce to replace the cells removed.
In contrast, other types of cell, such as brain cells, have long life spans and
are not replaced during a persons lifetime. If brain tissue is damaged, most cell
types in the brain cannot reproduce to replace the damaged cells.

KEY IDEAS

Cells were first identified and named by Hooke in 1665.

The nucleus in a cell was identified and named by Brown in 1831.
The Cell Theory arose in the mid-1800s.
The Cell Theory recognises that all living things are composed of one or
more cells and that new cells are produced by existing cells.
The life span of cells in a multicellular organism varies.
The unit of measure often used in relation to cell size is the micrometre (Mm).

QUICK-CHECK
1 Suggest why cells were not discovered by the Greek physician Hippocrates
(died 357 BC).
2 Who is credited with the discovery of the basic building block of living
organisms?
3 Who is credited with the discovery of the cell nucleus?
4 What was the important contribution by Schleiden and Schwann to biology?
5 Identify one commonplace idea about the origin of living things before
Virchow.
6 Which person is more likely to have permanent damage after an accident:
person A who survives after blood loss or person B who survives after some
loss of brain tissue? Explain.
7 How many micrometres (Mm) are there in a millimetre (mm)?

CELLS: DISCOVERY AND EXPLORATION

Tools for viewing cells

With few exceptions, individual cells typically are too small to be seen with an
unaided eye. Because of this, the study of cells has depended on the use of instruments, such as microscopes. There are many different kinds of microscopes but
they can be broadly divided into two groups, light and electron.

Light microscopes
Some microscopes used for viewing
a dissection or a small organism
use light being reflected from the
surface of the organism.

Hooke needed a light microscope to see the dead cells present in cork. Light
microscopes (LMs) increase the ability of the human eye to see tiny objects.
LMs can reveal objects such as the unicellular organism in figure 1.11a that
are too small to be seen, or details that are too minute to be resolved with an
unaided human eye. LMs use visible light that illuminates and passes through a
specimen. When tissues are examined using an LM, a slice of tissue just a few
cells thick is viewed. The tissue is usually stained with a dye (see page 11) to
make it more visible.

Simple light microscope

ODD FACT
In the 1990s,
a new technique, known
as near-field scanning optical
microscopy (NSOM), was
developed for viewing cells
and other objects. The
technique allows organelles
that are too small to be resolved
with a normal light microscope
to be seen.

Figure 1.9 An example

of a compound light microscope

10

NATURE OF BIOLOGY BOOK 1

Light microscopes (LM) use glass lenses. Early LMs with only one lens, like the
kind used by Hooke and van Leeuwenhoek, are called simple light microscopes.
They are similar to a magnifying glass.

Compound light microscope

Microscopes with at least two sets of lenses are called compound light microscopes (CLM). Most compound light microscopes have several objective lenses,
each of a different magnification (see figure 1.9). The amount of magnification
you obtain when using a light microscope, that is, how large the object appears,
depends on the magnification powers of both the objective lenses and eyepiece
(ocular) lenses you use. The magnification you obtain of an object is calculated by
multiplying the magnification (power) of
the objective lens by the magnification
Eyepiece lens (10x)
of the ocular lens you use.
The highest magnifications are
obtained with the use of an oil immersion objective lens. Light usually
travels in a straight line through a particular medium. As light passes from one
medium to a different medium, the rays
change direction they are refracted.
Hence, as light passes through a glass
slide holding a specimen and into air
above, rays are refracted and there is a
reduction of light entering the objective
Objective lens (10x)
lens (figure 1.10). A reduction of light
reduces the clarity of an image. With an
Objective lens (4x)
oil immersion objective lens, oil of the
same refractive index as glass is placed
Objective lens (40x)
between the glass slide and the objective
lens. This reduces the loss of light due
to refraction and higher magnifications
are possible. Oil immersion lenses
usually have a magnification of 100,
compared with the usual maximum of
40 with a dry lens.

Figure 1.10 Note that the use of oil (with the same
refractive index as glass) between the specimen and
the objective lens increases the amount of light passing
through the optical system of the microscope by reducing
refraction. Higher magnification objective lenses can be
used and a clearer image of the specimen is obtained.

As light moves
from glass into
air it is refracted
away from the
vertical and hence
away from the
objective lens

Objective

Immersion oil
Air

Glass
slide

Condenser
lens
Light

Figure 1.11 (a) Image of Paramecium, a unicellular organism,

as seen with a light microscope (b) Same type of cell as seen with
a phase contrast microscope

Characteristics of the lenses also influence a microscopes

resolution. Resolution is the ability to see two points that are
close together as two separate points. Our eyes have limited
resolving power: they may interpret two small spots that are
close together as a single blurred spot. We use microscopes to
resolve things that our eyes are unable to see; with an appropriate microscope we can distinguish the two small spots. But
microscopes also have a limit to their resolving power. A poor
quality microscope might simply magnify the blur we see into
a larger blur. The wavelength of light used, as well as the characteristics of the lenses, influence the size of an object that can
be resolved with a microscope. The smaller the wavelength of
light used, the smaller the size discernible. Standard light microscopes use visible light.
Cells are virtually colourless and hence are difficult to see
under a standard LM. Staining is required. Groups of cells are
also cut into thin slices before staining. These treatments necessarily kill cells and often distort cell features. During the
twentieth century, other kinds of microscopes and techniques
as described below were developed for viewing and analysing
cells.

Phase contrast microscope

The phase contrast microscope is a modified compound light
microscope (CLM) that was developed to observe unstained,
intact living cells (figure 1.11). These microscopes use the fact
that different parts of a cell transmit and change the direction of
light to varying degrees and enhance that difference. The image
produced has highly contrasting bright and dark areas.

Fluorescence microscope

Figure 1.12 Cancerous breast cells viewed with a

fluorescence microscope after staining for the presence of
vimentin (green) and keratin (red)

Another kind of CLM is the fluorescence microscope, which

uses ultraviolet (UV) light to reveal compounds that have been
stained with fluorescent dyes that bind to particular compounds
in a cell. The colour of fluorescence depends on the particular
fluorescent stain being used and the nature of the compound to
which it is attached (see figure 1.12).
CELLS: DISCOVERY AND EXPLORATION

11

Scanning confocal microscope

Another development has been the scanning confocal microscope (figure 1.14).
Image to computer screen
Eyepiece
lenses

Laser

Image to monitor
screen

Fluorescence
light source

Confocal
pinhole
Confocal
pinhole

Detector
Objective
Object
in focal plane
not in focal plane

Figure 1.13 In a confocal

microscope, light outside the focal
plane is excluded from the detector
by a pinhole aperture.

Figure 1.15 A Drosophila embryo

that has been laser scanned. The lefthand side shows selected optical slices
of the embryo. The right-hand side is
a projection of the entire 47 optical
slices.

12

NATURE OF BIOLOGY BOOK 1

Objective
lenses on
revolving
nosepiece
Stage
Condenser
Focusing knob
Filters and
diaphragm

Halogen
light
source for
transmitted
light

Figure 1.14 Confocal microscope note the parts that you recognise from the microscopes
you use in practical classes. Other features allow the microscope to be connected to computer
and video systems. Because of the detailed work possible, settings used often by an operator
can be stored in a computer and fed back to the microscope as required.
Confocal microscopy uses laser light and special optics
to allow a viewer to look at successively deeper layers
of an object, such as a cell or micro-organism, without
having to cut it into the many thin sections required by traditional light microscopy. Fluorescent stains are also used.
Fluorescence coming from the specimen is focused by
an objective lens through a pinhole aperture to a detector
(figure 1.13). Fluorescence from out-of-focus planes
above and below the in-focus plane is not transmitted. The
fact that out-of-focus images are not transmitted means that
the only image received by the detector is a sharply in-focus
image of a thin slice of specimen (see figure 1.12 on page 11).
The blurriness of out-of-focus parts is no longer observed.
Another advantage of confocal microscopy is that it can
be combined with scanning microscopy, in which a user can
perform 3-D microscopy of fluorescently labelled specimens or
reflective surfaces. A computer is used to digitise the image of
each section of the specimen obtained from confocal microscopy and the results are combined to give a 3-D image of the
specimen that can be rotated and viewed from various aspects
(see figure 1.15).
Microscopes are now commonly used in combination with computers and
automated cameras. Computers can analyse the shape, colour and density of
images seen under a microscope and enable biologists to easily carry out tasks
that would otherwise be too difficult or too time consuming (figure 1.16).
Biologists such as Associate Professor Leigh Ackland use microscopes and
techniques such as those described above and later in the chapter. Read what
Leigh writes about her work on page 13.

Figure 1.16 Confocal microscope

and associated equipment. Note the
glasses on the bench that are used for
3-D microscopy.

BIOLOGIST AT WORK
Associate Professor Leigh Ackland Molecular Biologist
Associate Professor Leigh Ackland is a Research Scientist and Senior Lecturer at the Centre for Cellular and
Molecular Biology, School of Biological and Chemical
Sciences, at Deakin University. Leigh writes:
Since my early days as a research biologist, I have been
very interested in studying the biology of cells by getting
them to grow outside the body, using tissue culture techniques. Recent knowledge of the nutritional requirements
of different types of cells has enabled biologists to grow
cells taken from parts of the body such as skin, gut, breast
and placenta. Using this approach, we have learned much
about the life of a cell. In culture, cells grow, become
specialised to carry out different functions, communicate
with each other and their environment and eventually die.
The study of cells in tissue culture has provided a
wealth of information about the behaviour of normal cells
and diseased cells, such as cancer cells. Cancer cells start
their life as normal cells but undergo changes causing them
to grow without control, to lose contact with each other
and with the substrate to which they are attached. These
changes can lead cells to spread around the body.
I became interested in finding out about how normal
breast cells turn into cancerous cells when I started
working with a human breast cancer cell line which had
the unusual capacity to develop into different subtypes of
cells. This line provided an opportunity for us to develop
a model of the human breast for studying cancer. Breast
cancer cells arise from the glandular part of the breast
which consists of epithelia (refer to figure 4.20, page
89). Different epithelial cells can be identified by the
types of structural proteins (cytoskeleton) they contain.
Together with members of my laboratory, I developed a
tissue culture model to represent the glandular structures
of the normal breast.
Using this model, we have shown that the normal
behaviour of the cells could be converted to the abnormal

Figure 1.17 Associate Professor Leigh Ackland. When cells

are not being used for experiments they can be frozen in liquid
nitrogen. First, an anti-freeze agent is added to the culture to
prevent damage to cell membranes.

behaviour characteristic of cancer cells. The converted

cells were not able to form proper contacts with each other
and with the extracellular environment. They showed other
changes, including alteration in expression of different
intracellular markers, in particular one cytoskeleton marker
called vimentin. Vimentin protein has been correlated with
the degree of invasiveness of the cancer.
Cancer is a very complex disease. Many research scientists are working on different aspects of it, ranging from
the role of the immune system and the role of cellular
microenvironment to the epidemiology of cancer. These
studies will all contribute to understanding how cancer
cells arise and what factors are important in the progression of the disease.
My interest in science was kindled by my maternal
grandfather, a chemistry teacher, who took me on expeditions to places like museums and questioned the
science behind what we saw. His inspiration stimulated
me to take science at school and then at university.

CELLS: DISCOVERY AND EXPLORATION

13

Electron microscopes
Transmission electron microscope
In the 1930s, the transmission electron microscope (TEM) was developed.
Instead of light, a beam of electrons with a much shorter wavelength passes
through and is used to illuminate specimens. Instead of glass lenses that control
the passage of light rays in LMs, a TEM has a series of electromagnets that each
create an electromagnetic field to control the path of the electron beam. Figure
1.18 shows a comparison between the internal structure of a light microscope and
a transmission electron microscope. Note the similarities; note the differences.
 




 
 


Figure 1.18 (a) Optical system of

a light microscope (LM). The light
source is visible light and glass
lenses (gl) produce an image that
can be detected by an eye or other
appropriate receptor such as a camera.
(b) Optical system of a transmission
electron microscope (TEM). A tungsten
filament emits a beam of electrons
which is controlled by a series of
electromagnetic lenses (el). In this
figure, the orientation of the TEM
system has been reversed to allow
direct comparison of its components
with those of a light microscope. In
reality, the filament is at the top and
the viewing screen at the bottom so
a TEM resembles an inverted light
microscope.









Intermediate lenses



Objective lenses

 




 
 


Specimens


Condenser lenses


 

 
 








TEMs have a much greater resolving power than light microscopes (see table
1.2) because of the short wavelengths of electron beams. TEMs have revealed the
presence of many kinds of cell organelles and have shown the complex internal
structure that exists within cells. (See pages 33 and 34, figures 2.14b and 2.16a,
which show part of the internal structure of a cell as seen with a TEM.)

Table 1.2 Comparison of the

human eye, light microscope (LM)
and transmission electron microscope
(TEM). The smaller the separation
distance between two objects, the
larger the resolving power of the
instrument being used.

Human eye

LM

TEM

smallest resolvable
separation distance

0.1 mm
(100 Mm)

0.000 2 mm
(0.2 Mm)

0.000 000 5 mm
(0.0005 Mm)

source of illumination

light rays

light rays

electron beam

Scanning electron microscope

The scanning electron microscope was released in 1965. This instrument is
able to provide detailed images of surfaces (see figure 1.19). An electron gun
produces an electron beam that is focused onto one spot on the surface of a
specimen and is then scanned back and forth along the specimens surface. The
surface releases another set of electrons from the specimen and these form an
image on a small fluorescent screen. Depending on their size, whole organisms
can be scanned (figure 1.19).

14

NATURE OF BIOLOGY BOOK 1

Figure 1.19 Scanning

(a)

electron micrograph of the pin

cushion millipede, Phryssonatus
novaehollandiae. Note (a)
the plates and hairs along the
dorsal surface and (b) the pairs
of legs and hairs visible on the
ventral surface of the same
organism. The adults of this
species grow to about 4 mm in
length and are abundant in the
sand and soils of Victoria.

mm = millimetre


m = micrometre
M
nm = nanometre

(b)

Although electron microscopes have greater resolving power than light microscopes, they can be used only with dead cells or organisms. The current ability of
modern light microscopes, such as the confocal microscope, to allow the detailed
study of living cells and identification and location of specific molecules in a
cell make light microscopes more appropriate for some settings in spite of their
reduced resolution. The size of the cell or organism under examination is also
important in the choice of instrument. Figure 1.20 outlines the limits of use of the
unaided eye, light microscopes and electron microscopes.






Human height
Length of some
nerve and
muscle cells e.g.
from giraffe neck
Chicken egg
Unaided
eye




A logarithmic scale is one in which

each marked unit moving up the
scale is 10 times larger than the
next. This contrasts with a linear
scale in which each marked unit is
the same size as the next.




Frog egg




Plant cells











Animal cells
Light
microscope

Nucleus
Mitochondrion
Bacteria

Viruses
Ribosome

Figure 1.20 The arrows on the

right-hand side indicate the ranges
over which viewing is possible with
an unaided eye, light microscope and
electron microscope. The logarithmic
scale indicates the size of organism,
cell or cell part visible by the
particular tools. 1 mm  1000 Mm;
1 Mm  1000 nm.




Electron
microscope

Proteins
Lipids






Small molecules

Atoms

CELLS: DISCOVERY AND EXPLORATION

15

Recent developments in
current systems
Light microscopes
Light microscopes have now been in use for centuries. As new technologies
and materials became available, the power and capabilities of microscopes have
changed significantly. Development continues. Computers have facilitated the
use of many microscopes. Other advancements include modification of existing
lens systems and automation in cells examination. We will consider two of these
recent technological advances.

Differential interference contrast (DIC)

microscopes
Differential interference contrast (DIC) microscopes (figure 1.21)
are used to obtain and examine three-dimensional impressions of an
object. The images are achieved using specially designed prisms to
split and then recombine the light. A recent modification involves
the way in which the light is split (the PlasDIC technique). This
has significantly improved the optical resolution and use of the
microscope.
The PlasDIC is a system that gives first-class, three-dimensional
views of an object. This is particularly important when fine-detailed
manipulation of living cells is required. One example of this is the
need to inject a sperm into the cytoplasm of an egg (oocyte) in some
cases of in vitro fertilisation (figure 1.22).
When a woman fails to conceive a child, it is sometimes due to
a low sperm count in the semen of the male. In such cases, fertilisation can sometimes be achieved by manually injecting a single
sperm into a mature egg. A good three-dimensional view of the
egg is essential to check that all parts of the egg are in good condition, hence ensuring the highest possible chance of success of the
injection.
Also in this modified optical system, plastic dishes can be used
instead of glass to hold any specimen. This is important, not only
because cells grow better in plastic receptacles than in glass, but
also because plastic equipment is far cheaper to use.

Figure 1.21 Differential

interference contrast microscope.
Note that the object being examined
is illuminated from above. This is
called an inverted system and is
important because it provides a
more stable system when manual
manipulation of an object is required.

Figure 1.22 Injection of a sperm

into the cytoplasm of an oocyte as
viewed with a Zeiss PlasDIC system

16

NATURE OF BIOLOGY BOOK 1

Automatic scanning of cells

In medical diagnosis, there are situations in which many thousands of cells must
be examined in a search for rare defective cells. Automatic scanning is now
possible using motorised scanning systems (figure 1.23).
The technique combines two powerful components. The first is a mechanised evaluation
platform holding the specimen slides. The platform
searches through and analyses the cells, up to 7000
per second, and singles out those that are defective.
The second powerful component is a computer that
stores the results of any highlighted defective cell,
as well as data related to the particular slide and
the position of the cell. This means that defective
cells can be readily found at a later time by simply
clicking the computer mouse.
The system operates with both single cells, such
as in a blood culture, and with groups of cells, for
example, cells in a section of breast tissue. Fluorescent stains are often used in such systems.

Figure 1.23 Automatic scanning of

up to 7000 cells per second is possible
with motorised scanning systems.
Rare defective cells are identified.
A computer records details about
each defective cell and its position
on the particular slide. A simple click
of a computer mouse allows reexamination of any defective cell.

Electron microscopes
Two other techniques are important for electron microscopy freeze fracture
and shadowing.

Freeze fracture
In freeze fracture, a small block of living or dead tissue is rapidly frozen in
liquid nitrogen. Virtually no change occurs to the molecules of the specimen
involved. The frozen tissue is placed into a vacuum chamber and broken with
the sharp edge of a knife. The fracturing of the specimen exposes internal
structures and their surfaces that can be examined after further treatment (see
figure 2.20c, page 37).

Shadowing

Figure 1.24 The technique of

shadowing occurs in a vacuum
chamber.

In the shadowing technique, fractured pieces of specimen are exposed to, and
partly covered by, heavy metal such as platinum or gold that is evaporated from
a heated wire to one side of the vacuum chamber. Because the metal atoms
come from one side of the chamber (see
Carbon
Vacuum
figure 1.24), the thickness of the metal film
electrode
chamber
reflects the contours of the parts that are
Metal evaporated
covered. The parts are then covered with a
from platinum wire
layer of carbon atoms that is transparent to
electrons. This layer strengthens the metal
replica. The specimen fragments are dissolved away leaving metal replicas that can
be examined with an electron microscope.
Shadow of
fragment on side
In this chapter we have examined a
uncoated with metal
range of tools and techniques important
in the study of cells, the basic structure of
all living things. Although there are basic
features that all cells share, there are also
Microscopic fragments
significant distinguishing features. We will
explore the structure and function of cells
of different kinds of organisms in the next
To vacuum
system
chapter.
CELLS: DISCOVERY AND EXPLORATION

17

KEY IDEAS
Various types of microscopes can be used to examine cells.
Light microscopes (LMs) reveal details about the arrangement of cells and
the internal structure of cells.
Compound light microscopes (CLMs) have at least two sets of lenses:
objective lenses and ocular (or eyepiece) lenses.
Cells are often stained with one or more dyes to make their various
components easier to see.
Phase contrast microscopes allow the study of unstained living cells.
Fluorescent microscopes reveal details of chemical substances present.
Confocal microscopes use lasers to produce a sharply in-focus image of a
thin layer of a specimen.
Electron microscopes use beams of electrons instead of beams of light.
Transmission electron microscopes (TEMs) reveal fine detail of the
internal structure of cells.
Scanning electron microscopes (SEMs) reveal details of cell surfaces.
Technological advances involving equipment and stains associated with
microscopy continue to be developed.

QUICKCHECK
8 If you had a choice of any kind of light microscope, identify, giving a
reason, the most appropriate one for viewing the following:
a a living amoeba
b a section of stained plant tissue
c the transfer of a nucleus from one cell into another.
9 True or false? Briefly explain your choice.
a All kinds of light microscopes use visible light to illuminate objects.
b If the objective lens of a light microscope has a 5 magnification and
its ocular lens is 10, then the magnification obtained of an object
being viewed is 15.
c The use of an oil immersion lens increases the magnification capability
of a microscope.
10 If you had a choice of any kind of electron microscope, identify, giving a
reason, the most appropriate one for viewing the following:
a the surface of a layer of cells
b a section of brain tissue
c a small insect about 1 mm long.
11 True or false? Briefly explain your choice.
a Electron microscopes can be used to view living and non-living
tissues.
b The resolving power of a TEM is greater than that of a LM.
c TEMs and SEMs are equally appropriate to use for viewing minute
organisms.

18

NATURE OF BIOLOGY BOOK 1

BIOCHALLENGE
1

2






 




 



Match these labels to the parts indicated on the diagram of a

compound light microscope:
objective lens eyepiece stage.



 


The magnifications are shown on these objective lenses and

eyepieces from a light microscope. What are the minimum
and maximum magnifications possible with these lenses?

What kind of light was used to obtain this image of cells from
an animal?

Given the detail in this image of part of a small animal,

what kind of microscope was used?

What kind of light was used to obtain this image of cells

from an animal?

A small microbe was placed on a microscope slide that had

a scale grid with lines at regular intervals of 10 m etched
into its surface. The microbe was examined with a light
microscope. The result was:

Given the dimensions of the grid, what is the diameter

of the microbe?

CELLS: DISCOVERY AND EXPLORATION

19

CHAPTER REVIEW
Key words
CROSSWORD

biogenesis
Cell Theory
cells
compound light
microscopes
eyepiece (ocular) lenses
fluorescence microscope
freeze fracture
light microscope
magnification

microscopes
nucleus
objective lenses
oil immersion objective
lens
phase contrast
microscope
resolution
scanning confocal
microscope

scanning electron
microscope
shadowing
simple light
microscopes
spontaneous generation
staining
transmission electron
microscope

Questions
1

Making connections The key words listed above can also be called
concepts. Concepts can be related to one another by using linking words
or phrases to form propositions. For example, the concept compound light
microscope can be linked to the concept lenses by the linking phrase
contains at least two to form a proposition. An arrow shows the sense of the
relationship:
contains at least two

compound
light microscope

lenses

When several concepts are related in a meaningful way, a concept map is

formed. Because concepts can be related in many different ways, there is no
single, correct concept map. Figure 1.25 shows one concept map containing
some of the key words and other terms from this chapter.
are made of

Special
glass

Lens/es
has only one
has at
least two

Visible
light

Simple
microscope

Compound
microscope

can be
can be

Light
microscope

uses
uses

has shorter
wavelength than

Ultraviolet
light

can be

Figure 1.25 Example of a

concept map

20

NATURE OF BIOLOGY BOOK 1

Microscope

can be

Electron
microscope

Use at least eight of the key words, and other words of your choice, to
make a concept map relating to microscopes and the contribution they have
made to the development of the Cell Theory and our ability to examine
cells.
Apply your understanding You wish to examine a number of specimens.
Refer to figures 1.4 (page 4) and 1.20 (page 15). Which microscope would
you use to examine each of the following?
a the surface of a cell membrane
b a frog egg
c a clear view of cytoskeletal fibrils in a cell
d a general overall view of a plant cell
e very small structures in cell cytoplasm
Using scientific terminology/conventions
When cells are being examined, dimensions are generally given in terms
of micrometre (m). It is important that you try to gain some understanding of how this measure relates to measurements of length that you are
already familiar with, measurements such as metre (m), centimetre (cm) and
millimetre (mm). Questions 3 and 4 are designed to give you practice at
understanding these comparisons.
a How many millimetres (mm) are there in a metre (m)?
b How many times larger than a millimetre (mm) is a metre (m)?
c How many micrometres (m) are there in a millimetre (mm)?
d How many times larger than a micrometre (m) is a millimetre (mm)?
Fill in the following blanks.
 ________ nm
a 1 Mm
b 1 ________  109 m
 ________ m
c 1 Mm
Communicating ideas Explain why using an oil immersion objective lens
has advantages over objective lenses that are used without the application of
oil.
Interpreting and communicating information using the Web Go to
www.jaconline.com.au/natureofbiology/natbiol1-3e and click on the
Microscopy weblink for this chapter. This information on microscopy
consists of four pages. After reading page 1, go to page 2.
a Compare the image obtained with a confocal microscope with a wide-field
image obtained with a standard microscope. Describe the difference(s)
between the two images shown on page 2.
b What is the key feature of confocal microscopy that results in a sharp
image being observed?
Now go to page 3 on fluorescence microscopy.
c What are the three components necessary for successful fluorescence
microscopy?
d What were the colours resulting from the use of the triple stain?
e What components did the triple stain reveal as being present in the cell?
Now go to page 4.
f Indicate one situation in which you might choose to use differential interference contrast rather than phase contrast microscopy.
You may wish to browse through other relevant sections of this website.

CELLS: DISCOVERY AND EXPLORATION

21