Scientific methodology
How to study the cell
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Visualisation of cell images
1. Nothing can be done without microscope
2. Organelles isolation
3. A panoramic view of the cell
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The definition of a “Cell”
The cell is the simplest collection of matter that can live
Although cells in multicellular organism (plants, animals etc.)
can not survive for long on their own they are basic units of
structure and function
All cells interact with the environment:
- they sense and respond to environmental changes;
- as open systems, they exchange materials and energy with
their surroundings.
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Microscopes are tools for study of cells
Discovered in 17th century, microscopes are in constant use
till now.
The most common is a light microscope (LMS)
Major characteristics are:
- magnification – the enlargement of the objective;
- resolution – clarity of the image
(minimum distance between two points which can be
separated and distinguished as two separate points).
LMS can rich the magnification of 1000x and resolution as
fine as 0.2 M, the size of a small bacterium.
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Nikon
dissecting
microscope
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Nikon
compound
microscope
Diopter
ring
Coarse
focus
knob
Fine focus
knob
Condenser
focus
Brightness
controll
ON/OFF
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Eyepiece
Objective
lenses
Mechanical
stage
Stage
adjustment
sCondenser
Daylight
filter
The relative size of living organisms
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The relative size of living organisms
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Types of light microscopy
Brightfield (unstained specimen):
light is directly passed through, the contrast is minor
Brightfield (stained specimen):
Enhanced contrast due to staining with various dyes
Fluorescence: shows the location of specific
fluorescently labelled molecules which absorb the
UV and emit visible light
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Types of light microscopy
Phase-contrast: variations in density within unstained
specimen enhance contrast, useful for examining of living
cells
Differential-interference-contrast (Nomarski): like p-c but
uses optical modifications to exaggerate differences in
density
Confocal: “optical sectioning” with lasers for imaging
particular region within a narrow depth of focus
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Electron microscope
EM focuses a beam of of electrons through the specimen.
Resolving power is inversely related to the wavelength of
radiation a microscope uses.
Electron beams have wavelength much shorter than visible
light. This helps the EM to have resolving power of about 2
nm.
Most subcellular structures (organelles) can not be
visualised by LM, cell ultrastructure is studied with the use of
EM.
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TEM
TEM – transmission electron microscope is similar to LM but
instead transmits electrons (light in LM) through
electromagnets (lenses in case of LM).
The image can be focused either onto a screen or onto
photographic film.
The use – mainly for the study of internal ultrastructure of
cells.
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Lily Parenchyma Cell (cross-section) (TEM x7,210).
This image is copyright of Dennis Kunkel
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SEM
SEM – scanning electron microscope.
The electron beam scans the surface of the sample, which is
coated with a thin film of gold.
The beam excites electrons on the sample’s surface, and the
secondary electrons are collected and focused onto a
screen.
This results in the appearance of three-dimensional image.
The use – detailed study of the surface of the object.
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Xylem
Conductive Vessel Element in Mountain Mahogany Wood
(SEM x750). This image is copyright Dennis Kunkel.
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Human Red Blood Cells, Platelets and T-lymphocyte
(SEM x 9,900). This image is copyright of Dennis Kunkel
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LM versus EM
LM advantageous for the study of live cells, it is cheaper in
use and requires less skills to operate
EM has much greater resolution and allows visualisation of
many organelles that are impossible to observe with LM but;
the organism has to be killed.
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Cell fractionation
Cell fractionation is the separation of the major organells in
order to study their individual function.
It requires: homogenization of the tissue, disruption of cell
structure and the separation of organells via various types of
centrifugation.
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Cell fractionation
Differential
Centrifugation
Homogenization
800 g
10 min
Tissue
Homogenate
cells
20 000 g
15 min
Supernatant
Pellet enriched
in nuclei and
cellular debris
100 000 g
60 min
150 000 g
3 hrs
Pellet enriched Pellet enriched
in mitochondria in “microsomes”
Pellet
enriched in
ribosomes
“Microsomes”
are pieces of plasma membranes and cells’ internal membranes
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A panoramic view of the cell
All organisms belong to either of two types of cells:
- prokaryotic;
- eukaryotic.
The major difference is the existence of the nucleus:
Pro (before) karyon (nucleus)
Eu (true) karyon (nucleus)
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Prokaryotic cells
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Eukaryotic cell
nucleus
cell membrane
rough ER
smooth ER
mitochondrion
Golgi complex
lysosome
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Prokaryotic and eukaryotic cells
•Streptococcus pyogenes, the bacterium that causes strep
throat, is an example of prokaryotes.
•Yeast, the organism that makes bread rise and beer
ferment, is an example of unicellular eukaryotes.
Humans, of course, are an example of multicellular
eukaryotes.
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Why are most cells microscopic?
With the increase to 5
units, the ratio of surface
area to volume
decreases
Rates of chemical
exchange with the
extracellular environment
is insufficient to maintain
the cell.
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Why are most cells microscopic?
(c) By dividing the large
cell into many smaller
cells, we can restore a
surface-area-to-volume
ratio
Larger organisms do not generally have larger cells than smaller
organisms, but more cells.
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Summary
The cell is the simplest collection of matter that can live
All cells interact with the environment:
- they sense and respond to environmental changes;
- as open systems, they exchange materials and energy with
their surroundings.
LMS
Major characteristics are:
- magnification – the enlargement of the objective;
- resolution – clarity of the image (minimum distance
between two points which can be separated and
distinguished as two separate points).
7/17/2016
Types of light microscopy
Brightfield:
light is directly passed through, the contrast is minor
Fluorescence:
shows the location of specific fluorescent labelled molecule
which absorb the UV and emit visible light
Phase-contrast: amplification of variations in density within
unstained specimen enhances contrast (examining of living
cells)
Differential-interference-contrast (Nomarski): like p-c but
uses optical modifications to exaggerate differences in
density
Confocal: “optical sectioning” with lasers for imaging
particular region within a narrow depth of focus
7/17/2016
Electron microscope
EM focuses a beam of of electrons through the specimen,
cell ultrastructure is studied with the use of EM.
TEM – transmission electron microscope is similar to LM but
instead transmits electrons (light in LM) through
electromagnets (lenses in case of LM).
SEM – scanning electron microscope, the electron beam
scans the surface of the sample, which is coated with a thin
film of gold.
7/17/2016
Cell fractionation
Cell fractionation is the separation of the major organells in
order to study their individual function.
It requires:
homogenization of the tissue,
disruption of cell structure and
the separation of organells via various types of
centrifugation.
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Reading
Campbell et al. Biology. Ch. 1, 18-27; Ch. 6, 94-99
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