AREA OF STUDY 1 UNIT Cells in action - Jacaranda | Shop · Diplococcus pneumoniae (causes pneumonia) Treponema pallidum (spiral — causes ... three-dimensional details of a specimen

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Chapter 1 Cells: discovery and exploration

Chapter 2 Structure and function of cells

Chapter 3 Composition of cells

Chapter 4 Cell replication

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1AREA OF STUDY 1

Cells in action

2 NATURE OF BIOLOGY BOOK 1

1 Cells: discovery and exploration

KEY KNOWLEDGEThis chapter is designed to enable students to:

appreciate the historical development of microscopy techniques

investigate current and emerging technologies in light and electron microscopy

understand the importance of technological advances to our knowledge of life forms and cells.

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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 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.

HIPImage

HIP

SyCoP

CELLS: DISCOVERY AND EXPLORATION 3

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

(1855–1916), 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 con-

structions produced by intelligent life. Figure 1.2a

shows Lowell’s 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!

The Viking Lander carried instruments to test for the existence of living organ-

isms 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 moun-

tains 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 Labor-

atory 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 environ-

ments 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

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.

(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 Lowell’s observations being flawed?

(b)


by a membrane boundary. The strongest direct evidence for past or present extra-

terrestrial 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 tech-

nological advances that have contributed to our knowledge and understanding of

life forms, and their living compartments or cells.

Cells and microscopes: an introductionCells 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.

Cell type Example Size

animal frog egg

human egg (note the

relative size of sperm)

human white blood cell

human red blood cell

1500 Mm

200 Mm

25 Mm

8 Mm

fungus yeast cell 5 Mm

bacteria Staphylococcus

(causes infections

such as boils)

Diplococcus pneumoniae

(causes pneumonia)

Treponema pallidum

(spiral — causes

syphilis)

1 Mm

0.1 Mm

0.3 Mm wide

and

10 Mm long

plant epidermal leaf

cell

200–400 Mm

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.

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 man’s beard is about 200 Mm

(0.2 mm) wide. Typical bacterial cells

range from 0.1–1.5 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.


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.Table 1.1 Some important

milestones in the development of

microscopes

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 BC–AD 100 In China, the first recorded uses of optical lenses

AD 79 (excavated 1748)

People of Pompeii used glass-crystal lenses

1200–1250 Robert Grosseteste, Bishop of Lincoln, UK, made a primitive but functional magnifying glass.

1590sDutch lens-makers, Hans Janssen and his son Zacharias, used two lenses to develop the first compound microscope, called ‘telescope’ by some writers.

1605–1619Cornelius Drebbel (1572–1633), a Dutch/English inventor of many scientific instruments, developed a machine for grinding lenses and improved the quality of compound microscopes.

1605–1614Galileo Galilei (1564–1642), 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.

1665Englishman Robert Hooke (1635–1703) published Micrographia. He describes ‘cells’ in a piece of cork (page 6 and figure 1.5) and draws many cell types. Hooke’s microscope could magnify 14–42 times.

1674Antony van Leeuwenhoek (1632–1723), 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).

1733Englishman 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.

1738German Johann Lieberkuhn added a metal reflector to the microscope to increase light falling on a specimen.

1831Robert Brown (1773–1858), a Scottish botanist and naturalist, described the nucleus in orchid cells (figure 1.7, page 7 and page 8).

1838Two Germans, botanist Matthias Schleiden (1804–1881) and zoologist Theodor Schwann (1810–1882), 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

1878Germans Ernst Abby and Carl Zeiss produced improved oil-immersion microscope lenses that significantly increased the ability to magnify cells (figure 1.10, page 11).

1931–1933 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.

1938Dutch 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.

1969Scientists 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 three-dimensional specimens such as a cell (as in figure 1.15, page 12).

2003PlasDIC 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).

2004Laser 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.


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 character-

istics 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

Cells: an historical overviewIn his book, Micrographia, published in 1665, English scientist Robert Hooke

(1635–1703) 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,

Hooke’s observations were important because he was

the first to realise that this plant material had an organ-

ised structure at the microscopic level.

In 1674, a few years after Hooke discovered cells, a Dutch cloth merchant,

Anton van Leeuwenhoek (1632–1723), 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 Leeuwenhoek’s original interest with microscopes was

to examine fibres in the cloth he traded, he was inspired by the publication of

Hooke’s Micrographia.

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.

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?


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

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.

(a) (b)

(c)

(a) (b)


Leeuwenhoek built over 50 simple microscopes to examine material from dif-

ferent sources. Figure 1.6c shows Leeuwenhoek’s drawings of some of the algal and other cells he observed in pond water. More than 150 years later, in 1831, Robert Brown (1773–1858), 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 nucle-

ated 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 TheoryBy 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 (1804–1881), suggested that cells were the basic structural unit of all plant matter. A German zoologist, Theodor Schwann (1810–1882), 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, pub-

lished 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.

A German doctor, Rudolf Virchow (1821–1902) added to the under-

standing of cells by providing a new answer to the question: How are new

living things produced? Past answers to this question included spon-

taneous generation, the idea that living things could arise from non-

living 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:

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.

Figure 1.8 According to one theory,

living things could arise from dead

matter; for example, leaves could

become animals or fish, depending on

where they fell.

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’.


… 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.

Virchow’s contribution extended the Cell Theory to include the basic concept:

New cells are produced from existing cells.

In 1862, the French biologist, Louis Pasteur (1822–1895) carried out experi-

ments that conclusively disproved the old idea of spontaneous generation, and

supported the view that new cells are produced by existing cells.

Life span of cellsCells 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 2–3 days

• skin cells 20–35 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 person’s 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 person’s lifetime. If brain tissue is damaged, most cell

types in the brain cannot reproduce to replace the damaged cells.

ODD FACT

‘Spray-on skin’cells are now used in the treatment of some burns

(see chapter 4, page 77).

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).

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KEY IDEAS

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)?

QUICK-CHECK

The terms unicellular and

multicellular refer to organisms

built of one or more building

blocks respectively.


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.

Tools for viewing cellsWith 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 instru-

ments, such as microscopes. There are many different kinds of microscopes but

they can be broadly divided into two groups, light and electron.

Light microscopesHooke 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 microscopeLight 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 microscopeMicroscopes with at least two sets of lenses are called compound light micro-

scopes (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

of the ocular lens you use.

The highest magnifications are

obtained with the use of an oil immer-

sion objective lens. Light usually

travels in a straight line through a partic-

ular 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

lens (figure 1.10). A reduction of light

reduces the clarity of an image. With an

oil immersion objective lens, oil of the

same refractive index as glass is placed

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.

Some microscopes used for viewing

a dissection or a small organism

use light being reflected from the

surface of the organism.

Eyepiece lens (10x)

Objective lens (4x)

Objective lens (40x)

Objective lens (10x)

Figure 1.9 An example

of a compound light microscope


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.

Characteristics of the lenses also influence a microscope’s

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 appro-

priate 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 char-

acteristics 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 micro-

scopes 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 nec-

essarily 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 microscopeThe 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 microscopeAnother 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).

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

Immersion oil

Condenserlens

Glassslide

Air

Light

As light movesfrom glass intoair it is refractedaway from the vertical and henceaway from theobjective lens

Objective

Figure 1.12 Cancerous breast cells viewed with a

fluorescence microscope after staining for the presence of

vimentin (green) and keratin (red)


Scanning confocal microscopeAnother development has been the scanning confocal microscope (figure 1.14).

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 tra-

ditional 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 micros-

copy 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 1.15 A Drosophila embryo

that has been laser scanned. The left-

hand side shows selected optical slices

of the embryo. The right-hand side is

a projection of the entire 47 optical

slices.

Figure 1.13 In a confocal

microscope, light outside the focal

plane is excluded from the detector

by a pinhole aperture.

Laser

Confocalpinhole

Detector

Confocalpinhole

Objective

Objectin focal planenot in focal plane

Image to computer screen

Eyepiecelenses

Objectivelenses onrevolvingnosepiece

Stage

Condenser

Focusing knob

Filters and diaphragm

Image to monitor screen

Fluorescencelight source

Halogen lightsource for transmitted light


Figure 1.16 Confocal microscope

and associated equipment. Note the

glasses on the bench that are used for

3-D microscopy.

Associate Professor Leigh Ackland is a Research Sci-

entist 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 tech-

niques. 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

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 sci-

entists 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 progres-

sion of the disease.

‘My interest in science was kindled by my maternal

grandfather, a chemistry teacher, who took me on expe-

ditions 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.’

BIOLOGIST AT WORK

Associate Professor Leigh Ackland — Molecular Biologist

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.


Electron microscopesTransmission electron microscopeIn 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.

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.)

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 microscopeThe 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 specimen’s 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).

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.

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.

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Intermediate lenses

Objective lenses

Specimens

Condenser lenses


Although electron microscopes have greater resolving power than light micro-

scopes, 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.

Figure 1.19 Scanning

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.

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.

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.

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��◊ Length of some nerve and muscle cells e.g. from giraffe neck

◊ Chicken egg

◊ Frog egg

Plant cells

Animal cells

◊ Nucleus

Viruses

◊ Ribosome

Proteins

Lipids

Small molecules

◊ Atoms

◊ Mitochondrion◊ Bacteria

Unaidedeye

Lightmicroscope

Electronmicroscope

◊ Human height

(a) (b)

mm = millimetre

� ��m = micrometre

nm = nanometre

M


Recent developments in current systemsLight microscopesLight 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) microscopesDifferential 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, ferti-

lisation 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 con-

dition, 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 1.22 Injection of a sperm

into the cytoplasm of an oocyte as

viewed with a Zeiss PlasDIC system

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.


Carbonelectrode

Vacuum chamber

Metal evaporatedfrom platinum wire

‘Shadow’ of fragment on side uncoated with metal

Microscopic fragments

To vacuumsystem

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 re-

examination of any defective cell.

Automatic scanning of cellsIn 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 com-

ponents. 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. Fluor-

escent stains are often used in such systems.

Electron microscopesTwo other techniques are important for electron microscopy — freeze fracture

and shadowing.

Freeze fractureIn 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).

ShadowingIn 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

figure 1.24), the thickness of the metal film

reflects the contours of the parts that are

covered. The parts are then covered with a

layer of carbon atoms that is transparent to

electrons. This layer strengthens the metal

replica. The specimen fragments are dis-

solved away leaving metal replicas that can

be examined with an electron microscope.

In this chapter we have examined a

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

significant distinguishing features. We will

explore the structure and function of cells

of different kinds of organisms in the next

chapter.

Figure 1.24 The technique of

shadowing occurs in a vacuum

chamber.


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.

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•

•

KEY IDEAS

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 tissuec 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 cellsb a section of brain tissuec 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.

QUICK–CHECK

BIOCHALLENGE


A small microbe was placed on a microscope slide that had

a scale grid with lines at regular intervals of 10 μm etched

into its surface. The microbe was examined with a light

microscope. The result was:

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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?

Given the dimensions of the grid, what is the diameter

of the microbe?

1 2

3 4

5 6

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CHAPTER REVIEW


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

Key words

CROSSWORD

Questions

1 Making connections ³ 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:

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.

Figure 1.25 Example of a

concept map

compoundlight microscope

contains at least twolenses

Lens/es

Simplemicroscope

Visiblelight

Compoundmicroscope

Electronmicroscope

Lightmicroscope

Ultravioletlight

Microscope

Specialglassare made of

has only one

can be

can be

uses

uses has shorterwavelength than

can be

can be

has atleast two


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.

2 Apply your understanding ³ 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 ³ When cells are being examined, dimensions are generally given in terms

of micrometre (μm). It is important that you try to gain some understand-

ing 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.

3 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 (μm) are there in a millimetre (mm)?

d How many times larger than a micrometre (μm) is a millimetre (mm)?

4 Fill in the following blanks.

a 1 Mm � ________ nm

b 1 ________ � 10–9 m

c 1 Mm � ________ m

5 Communicating ideas ³ Explain why using an oil immersion objective lens

has advantages over objective lenses that are used without the application of

oil.

6 Interpreting and communicating information using the Web ³ 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 inter-

ference contrast rather than phase contrast microscopy.

You may wish to browse through other relevant sections of this website.

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