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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 cellsDiscovered 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

Eyepiece

Objective lenses

Mechanical stage

Stage adjustmentsCondenser

Daylight filterBrightness

controll ON/OFF

Condenser focus

Fine focus knob

Coarse focus knob

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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 microscopyPhase-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

Homogenization

Tissue cells

Homogenate

800 g10 min

20 000 g15 min

Supernatant

Pellet enriched in nuclei and cellular debris

100 000 g60 min

Pellet enriched in mitochondria

150 000 g3 hrs

Pellet enriched in “microsomes”

Pellet enriched in ribosomes

“Microsomes” are pieces of plasma membranes and cells’ internal membranes

Differential Centrifugation

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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

cell membrane

mitochondrion Golgi complex

lysosome

smooth ER

rough ER

nucleus

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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?

Rates of chemical exchange with the extracellular environment is insufficient to maintain the cell.

With the increase to 5 units, the ratio of surface area to volume decreases

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Why are most cells microscopic?

Larger organisms do not generally have larger cells than smaller organisms, but more cells.

(c) By dividing the large cell into many smaller cells, we can restore a surface-area-to-volume ratio

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SummaryThe 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.

LMSMajor 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).

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Types of light microscopyBrightfield: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 densityConfocal: “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, 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.

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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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Reading

Campbell et al. Biology. Ch. 1, 18-27; Ch. 6, 94-99