Cell Biology

S. Rahgozar,PhD

University of Isfahan

Faculty of Science

1. An overview of cells and cell research

1.2. General methods of Cell Biology

1392-93

Robert Hooke

(1665)

The cellular

structure of cork

Light microscope

1670s: Antony van Leeuwenhoek

observed sperm

RBCs

Bacteria

1838: Schleiden & Theodor

proposed the “cell theory”

All organisms are composed of cells

Cells are not formed de novo

Microscope

Magnification

Main concerns

Resolution

1000

0.22 µm

λ = Wavelength (fo visible light: 0.4-0.7μm)

NA= Numerical Aperture

(The light gathering power)

= ŋ sinα

ŋ= refractive index of the medium

through which light travels

α= The angle corresponding to half the

width of the cone of light collected

by the lens

(1< ŋ <1.4)

Different types of light microscopy

Bright field microscopy

Contrast resulting from the absorption of visible light by cell components

Staining: Dye may react with proteins/nucleic acids

and increase the contrast

Bright field micrograph of stained

tissue

o Requires fixation which kills the

cells

Probing with:

Nucleic acid probes

Antibodies

Labeled with a variety of tags

Phase contrast microscopy

Bright-field microscopy

Phase contrast microscopy

Converts difference in density between

different parts of the cell to difference in

contrast using optical system.

Difference in density difference in phase

difference in contrast

Differential interference -

contrast microscopy

Differential interfrence contrast microscopy

Video enhanced differential

interference - contrast microscopy

Fluorescence microscopy

Studying the intracellular distribution of molecules

For fixed or living cells

Fluorescent micrograph of a lung cell

in which the DNA is stained blue and

microtubules green.

The use of green fluorescent protein (GFP)

A mouse neuron in which microtubule-associated

protein is fused to GFP and visualized by fluorescence

microscopy.

GFP (from jelly fish) is fused to a protein of

interest using recombinant DNA. The GFP –

tagged protein can then be expressed in cell

and detected .

Fluorescence recovery after

photobleaching (FRAP)

To follow the movement of GFP-labeled

proteins within living cells

Fluorescence resonance energy

transfer (FRET)

Confocal microscopy

A human cell in which microtubules and actin filaments are

stained with red and green fluorescent dyes Only in-focus light emitted from the chosen

depth of the specimen is detected

A series of images obtained at different depths can

be used to reconstruct a three-dimensional image

of the sample

Electron microscopy

A Positively stained WBC

Negatively stained actin filaments

Greater resolution (2-0.2nm)

shorter wavelength of electrons ie: 0.004nm

Transmission electron microscopy

Fixation, Staining with salts of heavy metals providing contrast by

scattering electrons.

Isolated organelle is deposited on a film and

metal stains around its surface. Specimens are cut into thin sections

Metal shadowing of actin/myosin

o Specimen is coated with a thin

layer of evaporated metal.

Surfaces that face the metal are

coated more heavily.

o Freeze fracture splits the lipid

bilayer, leaving proteins

embedded in the membrane

associated with one of the two

membrane halves

Freeze fracture Specimens are frozen at -196 ̊C, fractured with a

knife blade, then shadowed with platinum

Scanning electron microscopy

The surface of the cell is coated

with a heavy metal, and a beam of

electrons is used to scan across the

specimen.

Electrons that are scattered from

the sample surface are collected to

generate a 3 dimensional image

Subcellular Fractionation

o Sonication

o Grinding in a mechanical homogenizer

o Treatment with a high speed blender

Differential centrifugation

Density gradient centrifugation

Organelles are separated by

sedimentation through a gradient

of a dense substance.

Velocity centrifugation

Separation of organelles such as mitochondria,

lysosomes, peroxisome

Equilibrium centrifugation

Particles are centrifuged until they

reach an equilibrium position at

which their density is equal to that of

the high concentration surrounding

sucrose or cesium chloride solution.

Separation of different types of membranes or

macromolecules that are labeled with different

isotopes.

Growth of cells in culture

Complications

Long division time (~20 h)

Unidentified components of media

Amino acids, vitamins, growth factors

Normal cells stop growing after

certain divisions.

o Cell lines are

embryonic stem cells

or

cells derived from tumors

which may proliferate indefinitely

in culture.

Cultures of Animal and Plant cells

o Growth factors in animal cultures are polypeptides,

however,

growth factors in plant cells are small molecules which

may pass through the plant cell wall

o Embryonic stem cells in animal cultures

and

Callus in plant cultures

are undifferentiated cells which are capable of

forming any of the different cell types

Callus

Viruses

Human papilomavirus particles

Simple systems that can be used to

investigate the functions of cells