BIOL2146 CELL STRUCTURE & FUNCTION

WEEK 1
CELLS: HOW CAN WE STUDY THEM?
A/PROF DONALD WLODKOWIC
CELLS ARE VERY SMALL
HOW CAN WE STUDY THEM TO REVEAL STRUCTURE & FUNCTION

Source: cellimagelibrary.org
CELLS CAN BE ISOLATED
CELLS CAN REMOVED FROM TISSUES AND GROWN IN LABS

Source: cloud-clone.com
CELL CULTURE
TECHNIQUES ENABLING IN VITRO GROWTH OF CELLS

Source: freenewsman.com
Source: UNSW Cell Biology
IN VITRO CELL CULTURE
MULTIPLE EXPERIMENTS CAN BE CARRIED ON CELLS

Source: oboeipr.com

Examples of in vitro experiments

• how cells respond to drugs
• how cells proliferate
• how cells move and migrate
• genetic modifications of cells
• how cell communicate
Source: unisense.com
MICROCOPY: SEEING CELLS
STAPLE OF CELL BIOLOGY TECHNIQUES

Source: azooptics.com
MICROCOPY
STAPLE OF CELL BIOLOGY TECHNIQUES
The optical microscope, also called the light
microscope, is the oldest type of microscope which
uses visible light and lenses in order to magnify
images of very small samples.

It is a standard tool frequently used in biology.

The first basic optical microscopes were developed

in the 17th century.

Today, there are many variations available that

enable high level resolutions and sample contrasts.
With the help of computer-aided optical design and
automated grinding methodology, image quality has
improved immensely with negligible deviation.

Source: azooptics.com
TYPES OF MICROCOPY
FROM LIGHT TO ELECTRON MICROSCOPY
CELL PREPARATION BEFORE MICROCOPY
CELLS NEED TO BE OFTEN FIXED, STAINED AND SECTIONED
DIFFERENT TYPES OF LIGHT MICROCOPY
EXPLOIT DIFFERENT PROPERTIES OF LIGHT
DIFFERENT TYPES OF LIGHT MICROCOPY
EXPLOIT DIFFERENT PROPERTIES OF LIGHT
SEEING CELLS IN COLOURS
FLUORESCENT MICROSCOPY

Source: cellimagelibrary.org
IN FLUORESCENT MICROSCOPY
MOLECULES HAVE TO STAINED WITH FLUORESCENT MARKERS

Source: rsif.royalsocietypublishing.org
 LIVING CELL FLUORESCENT MICROCOPY
ENABLES US TO STUDY LIVING CELLS IN CULTURE
Division of a human stem cell: Programmed cell death:
orange stain - mitochondria different colours are markers labelling
different phases of cell death
FLUORESCENT MICROCOPY
EXPLOITS UNIQUE CHARACTERISTICS OF FLUORESCENT MOLECULES
FLUORESCENT MICROCOPY
EMPLOYS A SET OF FILTERS FOR DETECTION OF SIGNALS
FLUORESCENT MICROCOPY
EMPLOYS A SET OF FILTERS FOR DETECTION OF SIGNALS
FLUORESCENT MICROCOPY
CAN LOCATE SPECIFIC MOLECULES AND STRUCTURES IN CELLS

Source: micro.magnet.fsu.edu
LIMITATIONS OF LIGHT MICROCOPY
CAN ONLY RESOLVE DETAILS 200 NM APART
LIMITATIONS OF LIGHT MICROSCOPY
DIFFRACTION OF LIGHT LIMITS THE RESOLUTION TO 200 NM
Optical microscopes have to overcome
a critical limit in optical resolution
caused by the diffraction of visible
light wavefronts when they pass via a
circular aperture at the rear focal plane of
an objective (lens).
The resolution of a microscope is
proportional to the size of its objective,
and inversely proportional to the
wavelength of light being observed.

The resolution of a microscope is not

controlled by the instrument’s quality but
by the wavelength of light used and the
aperture of its optics.
LIMITATIONS OF LIGHT MICROSCOPY
DIFFRACTION OF LIGHT LIMITS THE RESOLUTION TO 200 NM
An optical microscope cannot
resolve two objects located closer than
!/2NA, where:
• ! is the wavelength of light
• NA is the numerical aperture of the
imaging lens.

In other words, di"raction limits the

ability of the microscope to
distinguish between two objects
divided by a lateral distance of less
than half the wavelength of light
used to image the sample.
BREAKING THE DIFFRACTION LIMIT
SUPER-RESOLUTION FLUORESCENCE MICROCOPY
SUPER-RESOLUTION MICROSCOPY
NEXT GENERATION TOOL FOR CELL BIOLOGY
ELECTRON MICROSCOPY
SEEING IN NANOMETER SCALE USING ELECTRONS
TRANSMISSION ELECTRON MICROSCOPY
BEAM OF ELECTRONS IS TRANSMITTED THROUGH A SPECIMEN TO FORM AN IMAGE
SCANNING ELECTRON MICROSCOPY
BEAM OF ELECTRONS IS SCATTERED BY A SURFACE OF SPECIMENS
SCANNING ELECTRON MICROSCOPY
BEAM OF ELECTRONS IS SCATTERED BY A SURFACE OF SPECIMENS
NEXT GEN ELECTRON MICROSCOPY
ION ABRASION SCANNING ELECTRON MICROSCOPY
BEYOND MICROSCOPY
BIOCHEMICAL AND MOLECULAR TECHNIQUES
CELL FRACTIONATION
INDIVIDUAL CELL COMPARTMENTS CAN BE SEPARATED
Cell fractionation is the process used to separate cellular
components while preserving individual functions of each
component. This process is performed using di"erential
centrifugation
CELL FRACTIONATION
INCLUDES CELL HOMOGENISATION AND CENTRIFUGATION
Step 1 - Homogenisation
Tissue is typically homogenized in a bu"er solution that is isotonic to stop osmotic
damage. Mechanisms for homogenization include grinding, mincing, chopping,
pressure changes, osmotic shock, freeze-thawing, and ultra-sound. The samples are
then kept cold to prevent enzymatic damage. It is the formation of homogenous mass
of cells (cell homogenate or cell suspension). It involves grinding of cells in a suitable
medium in the presence of certain enzymes with correct pH, ionic composition, and
temperature. For example, pectinase which digests middle lamella among plant cells.

Step 2 - Di"erential centrifugation

The sequential increase in gravitational force results in the sequential separation of
organelles according to their density. After each centrifugation the pellet is removed
and the centrifugal force is increased. Finally, purification may be done through
equilibrium sedimentation, and the desired layer is extracted for further analysis.
CELL FRACTIONATION
DIFFERENTIAL CENTRIFUGATION
CELL FRACTIONATION
DIFFERENTIAL CENTRIFUGATION

In di"erential centrifugation,
you can isolate and enrich the
following cell components, in the
separating order:

• Whole cells and nuclei;

• Mitochondria, chloroplasts,
lysosomes, and peroxisomes;
• Microsomes (vesicles of disrupted
endoplasmic reticulum); and
• Ribosomes and cytosol.
PROTEIN SEPARATION
PROTEINS CAN BE SEPARATED USING CHROMATOGRAPHY
Column chromatography is frequently
used by to purify proteins. A sample of
water soluble mature of proteins is
loaded onto a column of adsorbant, such
as silica gel or alumina. A solvent
continuously flows down through the
column. Components of the sample
separate from each other by partitioning
between the stationary packing material
and the mobile solvent. Molecules with
di"erent chemical characteristics can be
fractionated and isolated from one
another because they move through the
column at di"erent rates.
 PROTEIN SEPARATION
PROTEINS CAN BE SEPARATED USING PAGE
Polyacrylamide gel electrophoresis
(PAGE), describes a technique widely
used in biochemistry, forensics,
genetics, molecular biology and
biotechnology to separate biological
macromolecules, usually proteins or
nucleic acids, according to their
electrophoretic mobility.

Mobility is a function of the length,

conformation and charge of the
molecule.
 PROTEIN SEPARATION
PROTEINS CAN BE SEPARATED USING PAGE

Molecules may be run in their native state, preserving the

molecules' higher-order structure. This method is called
Native-PAGE.

Alternatively, a chemical denaturant may be added to remove

this structure and turn the molecule into an unstructured
molecule whose mobility depends only on its length and mass-
to-charge ratio. This procedure is called SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE) is a method of separating molecules based on
the di"erence of their molecular weight.
DNA SEPARATION
DNA CAN ALSO BE SEPARATED USING GEL ELECTROPHORESIS
DNA electrophoresis is a common lab
technique used to identify, quantify, and purify
nucleic acid fragments.

Samples are loaded into wells of an agarose or

acrylamide gel and subjected to an electric field,
causing the negatively charged nucleic acids to
move toward the positive electrode.

Shorter DNA fragments with lower molecular

mass will travel more rapidly, whereas the
longest fragments with higher molecular mass
will remain closest to the origin of the gel,
resulting in separation based on size.
THANK YOU