Topic 2: Introduction to Cell Biology
Developments in microscopy and discoveries on cells and their structures gave birth to new
fields. One field that emerged was cytology. Cytology deals with the study of individual cells
while histology is the study of tissues. Cell biology combines both with biochemistry and
genetics. Its main focus is to give one an understanding on the structure and function of cells.
This topic focuses on introducing the cell to the student, as well as to show basic features we
see in all cells. The last part of the topic focuses on microscopy and how it has helped cell
biologists visualize cells under the microscope.
The field of cell biology
Cells are the basic unit of structure and function in all living things. They are considered as the
smallest unit of life. If one wants to understand living multicellular organisms, one has to first
understand the cell. All of the complex functions we see in multicellular organisms are due to
the actions of individual cells working together. Cells make up the structure of living
organisms. Cells are also responsible for how living organisms react to and interact with their
environment. No matter if it’s a single-celled bacterium or a multicellular plant or animal, cells
are complex structures and cell biology works to make sense of everything a cell is capable of.
Biochemistry studies the chemical processes that occur in living organisms. Molecular biology
deals largely with the molecules that are important in living organisms like nucleic acids and
proteins. Physiology studies the normal function of living organism. Genetics is the study of
genes and heredity. Cell biology combines all of this but puts them in the context of the cell.
Cell biology, simply put, is the study of cells. This includes their cellular structures, their
cellular processes and the systems they use in order to interact with other cells. Cell biology
integrates all the above mentioned fields and focuses on how biomolecules are used by the cell
in order to live, reproduce and perform all its other functions.
Figure 2.1. The three domains of life. All living organisms are classified under three domains,
Bacteria or Eubacteria, Archaea and Eukaryota. Species are classified under different domains
based on similarities of their genetic sequences. (Source: Alberts et al., 2007)
All living organisms are made up of cells and can be classified into three domains: Archaea
where ancient bacteria are classified, Eubacteria where modern bacteria are classified and
Eukaryota where all eukaryotic organisms fall under. Differences in the sequence of highly
conserved ribosomal RNA sequences and cell structure form the basis of classification into
these domains. Members of domain Archaea differ from members of domain Eubacteria and
Eukaryota in the composition of their plasma membrane. Their plasma membranes contain
branched hydrocarbon chains. In addition, their cell walls also lack peptidoglycan,
differentiating them from other Eubacteria. All these cells in different domains all have unique
features but they also have similarities
Characteristics common to all cells
1. All cells use the same linear chemical code to store their hereditary information.
Cells are very similar to computers. Computers make use of quantifiable units of
measurement for data called bytes. The amount of information for data in a
computer can be measured in bytes. Computers make use of several methods to
store data. From floppy disks to compact discs and eventually to USB drives,
computers are able to store a variety of information that we can make use of. Living
cells are similar to computers in that they also make use of information in the form
of double-stranded deoxyribonucleic acid (DNA).
All known living cells on Earth store hereditary information in the form of DNA
molecules. In the same way a computer uses 1s and 0s to code for information,
DNA makes use of four nitrogenous bases – adenine, cytosine, guanine and thymine
– which are then further simplified into the letters A, C, G and T respectively. All
the information any cell needs to survive and function are found in its DNA.
2. All cells make use of template polymerization in order to replicate their hereditary
information.
DNA is a double stranded structure. Each strand is composed of monomers called
deoxyribonucleotides (we’ll shorten it to simply nucleotide from here until the end
of the module). These nucleotides have a sugar attached to a nitrogenous base, as
well as a phosphate group, which links it to the next nucleotide. DNA is a repetitive
string of these monomers. New copies of DNA are not produced simply out of
nowhere. DNA is replicated by attaching nucleotides onto a template. This template
is a pre-existing DNA strand. The DNA double-helix can be separated into two
separate strands where one strand serves as the template for making a new copy of
that DNA molecule. Template polymerization is the way in which cells copy DNA
sequences in the living world. Cells are able to replicate all of their DNA with very
low errors. Systems exist so that the cells can maintain the integrity of the genetic
material that they contain. The details of this replication process will be touched on
further into the module.
3. All cells transform parts of their DNA into the same intermediate form.
Simply copying DNA is not enough for cells to perform all the functions they are
required to do. Cells should be able to store and then make use of the stored
information. When we say ‘expression’, we refer to when cells make use of the
information they have stored. Expression of the information in DNA leads to the
formation of two molecules – ribonucleic acid (RNA) and proteins. The process of
expression begins with template polymerization known as transcription where DNA
molecules are used as templates for the formation of shorter, single-stranded
polymers known as RNA. After RNA synthesis, these RNA molecules are further
transformed into protein through a process called translation. This module will
touch on the process of transcription in Unit 4.
4. All cells make use of proteins as catalysts.
Proteins are long polymers made of amino acid monomers. They make up a large
percent of the cell’s mass. Protein polypeptides are able to form structures with
reactive sites on their surface. These proteins bind with high specificity to molecules
and act as enzymes. These enzymes catalyse reactions that make or break bonds
between molecules. Proteins also maintain cell structures, they are responsible for
cell movement and they can sense a number of different signals that the cell needs
to survive. Proteins represent a part of the information present in DNA.
5. All cells translate RNA into proteins using the same process.
The information from DNA is first transcribed to RNA and then further changed
into proteins through a process called translation. Translation of RNA molecules
into proteins is similar across all living cells, with very few exceptions. Three bases
in RNA always correspond to one amino acid. Three kinds of RNA molecules
(messenger RNA, transfer RNA and ribosomal RNA) are essential components of
the protein translation machinery. Translation will also be covered in the later parts
of the module.
6. A portion of DNA that corresponds to one protein is one gene.
The hereditary information stored inside cells contains information for thousands
of proteins. Individual parts of DNA is transcribed into RNA molecules and then
translated into one protein. This individual part which corresponds to a single
protein is called a gene. There are many ways to process the RNA that comes out
from one gene. Differences in RNA processing leads to a diversity in the protein
product. This is why although DNA sequences are similar between humans, we can
observe very different characteristics of people from different continents in the
world.
Figure 2.2. Expression of proteins
from DNA sequences. Proteins are
produced by a process that firsts
consists of transcribing DNA into
RNA and then subsequently trans-
lating these RNA sequences into
proteins. Not all genes code for pro-
teins but all genes code for RNA
products. DNA contains all the
information an organism will need
throughout its lifetime. (Source:
Alberts et al., 2007)
7. All cells require free energy.
The cells use free energy in order to create and break covalent bonds. All this is
done in order to have a clear system in the cell. Replicating their DNA, transcribing
into RNA and then translating RNA into proteins all require the use of energy.
8. All cells are tiny factories that make use of the same basic building blocks.
Cells make use of the same biological molecules. DNA, RNA, proteins and other
macromolecules in the cells are composed of the same subunits. They make use of
the same types of sugars, lipids, and the same substances like adenosine
triphosphate (ATP). It is the combination of these molecules, and the polymers they
form, that contribute to different cells having very different characteristics from
each other.
9. All cells are surrounded by a plasma membrane that facilitates movement of nutrients
and waste materials across it.
All cells are enclosed by a plasma membrane. They differ in structure depending on
the species but all cells are enclosed by this plasma membrane. It acts as a barrier
that allows nutrients to enter the cell but does not allow the items synthesized in the
cell from easily leaking out. It ensures that intracellular processes are maintained
within the cell. Figure 2.3 shows electron micrographs of the plasma membranes of
different organisms.
Figure 2.3.Transmission electron micrographs of plasma membranes. All
living cells are enclosed by plasma membranes. Using electron microscopy, cell
biologists are able to visualize the lipid bilayer covering the cells. (A) E. coli plasma
membrane, PM: plasma membrane, PG: peptidoglycan, OM: outer membrane
(Source: Beveridge. (1999). J Bacteriol, 181(6):4725-33) (B) Plant model
organism A. thaliana plasma membrane (Source: Roger Innes, Indiana
University), (C) Small intestine epithelium (Source: Atlas of Plant and Animal
Histology)
10. A living cell can exist with just a few genes.
One of the organisms with the smallest known genome – the entirety of its genetic
material – is Mycoplasma genitalium. It lives using less than 500 genes which
encompasses 580,070 base pairs. Depending on the complexity of the cell, the
number of genes would also increase. Figure 2.4 compares the genome sizes of two
species of Mycoplasma.
Figure 2.4. Comparison of Mycoplasma pneumoniae and Mycoplasma
genitalium genomes. Different species have different genome sizes but their
genomes contain all the information the organism needs.
(Source: Himmelreich et al. (1997). Nucleic Acid Res, 25(4):701-712)
The microscope: The invisible world made visible
The microscope is one of the most important tools that help in the study of cell biology. By
combining the ability of the microscope to see microscopic structures with different staining
techniques that help in the visualization of said structures, cell biologists are able to see and
observe structures and processes in the cell.
Light microscopy is one of the most common techniques used in visualizing cells. There are
terms that we have to remember when we talk of microscopy. Magnification is the ratio of an
object’s image size to its real size. Resolution is a measure of clarity of the produced image.
Resolution can be defined as the minimum distance between two separate points wherein the
two points are still seen as two points. The light microscope has a limit of resolution equal to
0.2 micrometers or 200 nanometers. This is around the size of a small bacterium. The resolution
of the light microscope is limited by the shortest wavelength of light used to illuminate the
specimen. Another term that is mentioned frequently in microscopy is contrast. Contrast is the
difference in light intensity between the image and the adjacent background relative to the
overall background intensity. Good contrast makes it easier to differentiate parts in your
sample.
Normally, most bacteria and animal cells are colorless under the microscope. The illuminating
light passes through the sample and in the process, washes out the structures you see inside the
cell. This makes it difficult to observe intracellular structures. Originally, different kinds of
dyes were used as stains. These dyes directly stained all kinds of different biomolecules. There
were dyes for carbohydrates, stains for proteins and stains for nucleic acids among others.
There are now more targeted forms of staining which stain specific molecules in the cell and
will be discussed a little later in the chapter. Staining and labelling of cell components improve
contrast and allows one to see structures better.
Figure 2.5. Comparison of unstained and stained cells as viewed under
brightfield microscopy. (A) Unstained neutrophils, (B) Stained neutrophils, (C)
Unstained squamous epithelial cell, (D) Stained squamous epithelial cell. (Source:
Chu-su et al. (2017). Scientific Reports, 7:40521)
Many developments have been made in light microscopy that make it even easier to observe
cells under the microscope. As cell culture techniques developed, it became necessary to view
live cells in culture. The main problem they had with this was that light microscopes washed
out any features of these cells so there were very little things that could be seen. Most staining
methods at the time involved fixing the cells using chemicals like formaldehyde or acetic acid
and this killed the cells. In order to be able to still view the cells without the need for stains,
different modifications on the traditional light microscope were made.
Phase contrast microscopy and differential-interference-contrast microscopy (DIC) were
developed in order to observe living cells in culture. When light passes through a cell, the light
wave is changed according to the cells refractive index. Differences in thickness and density
throughout the cell create different effects on the passing light. Phase-contrast and DIC
microscopy make use of these changes in order to visualize cells and their internal structures
without the use of stains and cellular labels. Phase contrast microscopy results in bright halos
surrounding cells. Their intracellular structures are more defined than if using simple light
microscopy. DIC shows outer morphology of the sample so you would tend to see a three-
dimensional image using the microscope. Similar with phase contrast, intracellular structures
become more visible.
Another method developed for viewing cells was by observing the light scattered by its
components. A dark-field microscope directs illuminating rays of light towards the side so only
scattered light enters the microscope lenses. The result is that the cell looks bright against a
dark background. In normal brightfield microscopy, light passes directly though the sample
illuminating the entire field. Certain specimens were more visible using darkfield microscopy.
Similar to phase contrast microscopy, live, unstained samples are well suited for viewing using
this method. Darkfield microscopy requires only a few adjustments to the base machine.
Figure 2.6. Comparison of different light microscopy methods. Modifications on the traditional
light microscope makes it possible to view different structures better. There are many limitations to
traditional light microscopy and cell biologists continue to develop new imaging tools and
techniques in order to better view the cell in detail. (Source: Zeiss Campus)
Phase-contrast, differential-interference-contrast, and darkfield microscopy make it possible to
observe processes and events without killing the cell. Cell movements during mitosis and cell
migration can be observed using these techniques. A lot of these movements are very slow so
time-lapse movies are made. Cells are viewed at set time intervals and then these images are
brought together to form a sped up movie of the events being observed. These methods allowed
scientists to discover so many different things about living cells. Brightfield, phase-contrast,
DIC and darkfield microscopy all make use of transmitted light techniques to create images.
Most stains would stain all of a certain specific molecule. There are stains that target all
proteins, stains that target all nuclear DNA, stains that target starch plastids or stains that stain
all of the cytoplasm. Although there is much information that could be gathered through direct
staining, very little would be seen when we wanted to study say a specific protein like actin. In
order to further improve targeted visualization of cell components, scientists had to develop
new tools and techniques that made this possible.
When cell biologists want to see specific structures, they make use of fluorescence microscopy.
Think back on the electromagnetic spectrum (Figure 2.7.A). Visible light is composed of all
wavelengths from around 380 nm to 700 nm. Visible light is the part of the electromagnetic
spectrum that our eyes can see. Visible light is also used by the light microscope to illuminate
samples. Fluorescence microscopy makes use of the properties of visible light and the action
of fluorophores or what we call fluorescent molecules.
Figure 2.7. Fluorescence microscopy makes use of fluorophores that are excited by different
wavelengths of light. (A) The electromagnetic spectrum. (Source: da Silva et al. (2019). Aust J
Crop Sci, 13:1835-2707) (B) Diagram showing the difference between the excitation and emission
spectra of the fluorophores (Source: Horiba) (C) Different fluorophores have different spectra so
several fluorophores may be used to stain one sample. (Source: Cell Signaling Technology)
Fluorescent molecules absorb and are excited by light of a specific wavelength. In order to
return to a stable or ground state, these fluorescent molecules release the light energy absorbed
but at a different wavelength than the one absorbed (Figure 2.7.B). If we illuminate a
fluorescent molecule using the wavelength of light it absorbs, and then use a light filter which
only allows light of the emitted or released wavelength to be visible, what we see is a glow
against a dark background. The dark background allows even the smallest molecules to be
located and detected.
Traditional staining methods require a large amount of stain to make them visible under
brightfield microscopy. Fluorescence microscopy makes one able to see small molecules even
with very little of the fluorescent molecules present. These fluorescent dyes are viewed using
a fluorescence microscope. Its main difference from a traditional light microscope is the
presence of filters that allow only certain wavelengths of light to be detected by the microscope.
The first filter usually blocks out all wavelengths except the exciting wavelength and then the
second filter blocks everything except the emitting wavelength.
Figure 2.8. Visualization of
lamellipodia during cell move-
ment. The Rac pathway was ac-
tivated in 3T3 fibroblast cells and
the polymerizing actin filaments
were visualized. F-actin was
stained with green fluorescent
phalloidin staining while nuclei
were stained using DAPI.
(Source: Lao et al. (2016). J
Immunol, 196(2): 596-606)
Different fluorophores have different emission spectra (Figure 2.7.C). One use of this is that
different molecules can be targeted in a single sample. Figure 2.8 shows something like this.
The image shows a culture of 3T3 fibroblasts. 3T3 is a cell line that was established from albino
mouse embryonic tissue. The nuclear DNA was stained using DAPI (4′,6-diamidino-2-
phenylindole) which is a fluorescent dye that targets adenine and thymine rich regions in DNA.
DAPI is excited by wavelengths in the UV region (maximum absorption at 358 nm) of the
electromagnetic spectrum but emits a blue light (maximum emission at 461 nm). Filamentous
actin, one of the cytoskeletal elements of cells, was stained using phalloidin conjugated to a
fluorescent green fluorophore. Phalloidin is a toxin discovered from the poisonous mushroom
Amanita phalloides and it specifically binds to actin molecules in the cell. The fluorophore
emits light in the green spectrum (535-545 nm).
To get the image you see in Figure 2.8, the sample is first illuminated with UV light. The DAPI
filter will be used so only DAPI signals can be seen. After acquiring the image, without
changing the field, the illuminating light is changed to the one specific for excitation of the
green fluorophore. The filter is also changed so only the signal from the green fluorophore is
detected. The image is then acquired. When we create the image, we superimpose the images
taken using the DAPI filter and the green filter. The result is the image you see in Figure 2.8.
Proteins, nucleic acid sequences and other molecules in cells and tissues are the usual targets
of fluorescence microscopy. Fluorescent molecules are usually attached to antibodies specific
for the structure one wants to visualize. Antibodies are specific for their antigens so combining
fluorescent dyes with these antibodies allows one to locate specific molecules and structures.
For example, we want to stain the protein tubulin which is present in microtubules. We attach
a fluorescent dye to an antibody specific for tubulin. The antibody will attach to the tubulin
proteins in the cell and when the fluorescent dye is visualized, any place with the fluorescence
signal will be where the tubulin proteins are.
Figure 2.9. Diagram differentiating direct and indirect staining. Primary antibodies are specific
for the target while secondary antibodies are specific for the primary antibody. Indirect staining has
the advantage of making the observed signal brighter. These methods can also be applied to
enzyme-linked reporter assays. (Source: Cell Signaling Technology)
When we use antibodies as probes to detect and assay specific molecules in cells, the use either
direct or indirect staining. Direct staining or direct immunohistochemistry involves
attaching the fluorescent dye directly to the antibody targeting our desired molecule.
Unfortunately, sometimes it is hard to detect this fluorescent signal, either because there is too
little of the protein present or very little amount of antibody was able to penetrate and label the
sample. In order to solve this, indirect staining was developed. In indirect staining or indirect
immunohistochemistry, we usually amplify the fluorescent signal through chemical methods.
First, we make use of an antibody to recognize a specific target. This we call the primary
antibody. In order to observe a stronger signal, we usually attach another fluorescent dye to an
antibody specific for the primary antibody. This we call the secondary antibody. Primary
antibodies are usually monoclonal antibodies (recognize only one antigen) while polyclonal
antibodies (have more than one antigen) are usually used as secondary antibodies.
Fluorescent dyes are not the only thing that can be attached to antibodies. An enzyme can also
be used as a marker attached to the secondary antibody. Instead of fluorescence microscopy,
the marker is visualized by allowing the marker to break down a substrate leading to the
formation of a colored product. Common enzymes linked to antibodies are alkaline
phosphatase and horse radish peroxidase. These enzymes break down their specific substrates
and form colored precipitates. The location of the precipitate tells you the location of the
primary antibody. Radioisotopes can also be attached to these antibodies. Proper handling has
to be practiced when using them. The main advantage of using radioisotopes is that even the
smallest amount of the isotopes will be detected. This is used when there is very little amount
of the molecule you are targeting in the sample. Hormones, which are found in very small
amounts in the body, are usually detected using radioisotopes.
Figure 2.10. Different structures stained with different reporters. Depending on the
experiment, cell biologists make use of different reporters. Each type of reporter has an advantage
over the rest and much thought is put into how structures are to be visualized. (A) CD68 in mouse
liver sections stained using an HRP/DAB streptavidin-biotin ABC method. (Source: Jovicic et al.
(2015). PLoS ONE, 10(7):e1034089) (B) Staining of neurofilaments using FITC conjugated to anti-
2H3 antibodies. (C) Proteins labelled with radioactive leucine. (Source: Pittenger and Cleveland.
(1985). J Cell Biol, 101(5 Pt 1):1941-52)
Fluorescent dyes, fluorescent molecules, enzymes and radioisotopes are called reporters.
These reporters are visible and/or quantifiable items that can be used to measure or visualize
targets. These targets could be proteins, nucleic acids, sugars, or lipids. They may be attached
to antibodies for specific targeting of molecules or they can be attached to direct stains. Figure
2.10 shows examples of different reporters and how they would look like when viewed under
the microscope.
Figure 2.11. Comparison of images taken using a normal fluorescence
microscope and a confocal microscope. Confocal microscopy adds detail that is
lost using normal fluorescence microscopy. (A) Human medulla. (B) Whole rabbit
muscle fibers. (C) Grains. (Source: Olympus Life Science)
Figure 2.12. Confocal imaging of a
neuron. Confocal microscopy added depth
and more detail to samples being analysed
using fluorescence. The image contains
an intracellularly injec-ted layer III pyramidal
neuron of the human cingulate cortex. The
neuron’s processes are very distinct and we
can even determine spatial arrangement of
the processes coming from the soma.
(Source: Toharia et al. (2014).
Neuroinformatics, 12(2): 341-353)
Despite its many uses, fluorescence microscopy also has its limits. Specific structures are
difficult to resolve when there are numerous fluorescent signals located in one area. Confocal
microscopy was developed in order to overcome the limitations of traditional fluorescence
microscopy. Instead of illuminating the entire sample like they do in fluorescence microscopy,
confocal microscopy focuses a spot of light at a specific depth in the specimen. It makes use of
a bright pinpoint illumination, usually coming from a laser, whose light has passed through a
pinhole. The fluorescent signals are then collected by a detector. Complicated imaging software
then arranges the data being collected to produce a deconvoluted image of the specimen being
observed. An additional advantage of confocal microscopy is that it is not limited to the simple
X-Y plane of traditional microscopy. Confocal microscopy introduces a Z plane so we can
completely see all structures at different depths along the sample.
The limit of resolution of light microscopes makes it difficult to see small structures inside
cells. In order to increase the limit of resolution, scientists have developed electron
microscopes. These microscopes illuminate samples using electron beams instead of light rays.
Resolution is related to the wavelength of the illumination used in a microscope. Since electron
beams have much shorter wavelengths compared to visible light, they can achieve a resolution
of 0.002 nm given the wavelength of the electron beams. However, the optics system is unable
to properly perform some correction for aberrations of the imaging system so the electron
microscope’s limit of resolution is more around 0.1 nm. Despite this, it is still much lower than
light microscopes so makes it possible to view structures that would normally be unresolved
using traditional light microscopes.
There are two types of electron microscopes, the scanning electron microscope and
transmission electron microscope. Scanning electron microscopes (SEM) are useful when
studying the surface of samples. Samples are coated with a film of gold. Electron beams then
excite the electrons in the film and the microscope detects the excited electrons. The end result
is an image of the outer topography of the sample. The resulting image shows the sample in
three dimensions. Transmission electron microscopes (TEM) is used to visualize the internal
structure of samples. Electron beams are passed through the sample. Structures have been
stained with atoms of heavy metals so electrons which pass through the sample are scattered in
denser regions resulting in less electrons transmitted. The microscope detects the transmitted
electrons and the resulting image shows the internal structures of the specimen.
Figure 2.13. Comparing
of SEM and TEM. Yeast
cells (S. cerevisiae) were
viewed under both SEM
and TEM. SEM (A)
showed the outer mor-
phology of the yeasts
while TEM (B) showed
intracellular components.
(Source: Alberts et al.,
2007)
Electron microscopes are able to show structures which can not be seen using traditional light
microscopy. However, light microscopy is useful in observing living cells because samples in
electron microscopy have to be fixed and killed. Preparation of samples for electron
microscopy is also tedious and wrong preparation may result in the formation of artifacts that
can be seen in the image results.
Figure 2.14. Comparison of microscopy techniques. Scanning electron micrograph of
stereocilia projecting from hair cells found in the inner ear of a bullfrog (A) and images of the same
stereocilia using other techniques like differential interference contrast microscopy (B) and
transmission electron microscopy (C). (Source: Alberts et al., 2007)
Biological microscopes are the most useful tools of cytology, which is the study of cell
structure. Unfortunately, knowing the structure inside cells is not enough to understand how
they function. Modern cell biology combines cytology and biochemistry. This field makes
sense of the cellular structures we see by making use of biochemical assays and methods. The
combination of these two fields allows cell biologist to properly understand the structure and
function of cells.
