Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira
 MOLECULAR CELL BIOLOGY
 SIXTH EDITION
CHAPTER 9
Visualizing, Fractionating,
and Culturing Cells
©
2008 W.
H.©Freeman
andand
Company
Copyright
2008
W. H. Freeman
Company
Fluorescence
Alpha tubulin (green)
Actin (red)
DNA (blue)
Golgi (yellow)
Mitochondria (purple)
Cell Isolation,
Culture and
Differentiation
Proteases
•trypsin
Divalent cation chelator
•EDTA
Senescence
•50 times
Cell Strain: line of cell
that have a limited life
span
Cell line: line of cells that
are immortal
Media and Control
Controlled Factors:
•temperature
•atmosphere (pressure)
•humidity
•pH
•Ionic Strength
•Nutrients
• essential amino acids
• vitamins, salts, fatty acids, glucose, serum
•Antibiotics
Flow Cytometry
Hybridomas to produce monoclonal antibodies to specific proteins
•Myeloma cell: Immortal
•Mouse spleen cell: mortal
•Viral glycoproteins/polyethylene glycol
•Grown in media that does not allow
unfused cells to grow (mutations in
metabolic pathways). Fused cells have
ability to grow due to sharing of genetic
material that was deleted from other cells.
Left: Bright Field
Middle: Differential interference contrast
Right: Phase Contrast
The table below compares the different types of microscope:
http://universe-review.ca/R11-13-microscopes.htm
Characteristic
Compound Microscope
Transmission E. Microscope
Scanning E. Microscope
Resolution (Average)
500 nm
10 nm
2 nm
Resolution (Special)
100 nm
0.5 nm
0.2 nm
up to 1,500X
up to 5,000,000X
~ 100,000X
Depth of Field
poor
moderate
high
Type of Objects
living or non-living
non-living
non-living
Preparation Technique
usually simple
skilled
easy
Preparation Thickness
rather thick
very thin
variable
Specimen Mounting
glass slides
thin films on copper grids
aluminum stubs
large enough
limited
large
visible light
electrons
electrons
air
vacuum
vacuum
glass
1 electrostatic + a few em. lenses
1 electrostatic + a few em. lenses
mechanical
current in the objective lens coil
current in the objective lens coil
Magnification Adjustments
changing objectives
current in the projector lens coil
current in the projector lens coil
Specimen Contrast
by light absorption
by electron scattering
by electron scattering
Magnifying Power
Field of View
Source of Radiation
Medium
Nature of Lenses
Focusing
Compound
Compound microscopes are light
illuminated. The image seen with this
type of microscope is two dimensional.
Description This microscope is the most commonly
used. You can view individual cells, even
living ones. It has high magnification.
However, it has a low resolution.
Costs
Source of Radiation for
Image Formation
Medium
Specimen mounting
Nature of Lenses
Focusing
Magnification
Adjustments
Major Means of
Providing specimen
Contrast
Dissection or Stereoscope
Confocal Microscope
Scanning Electron Microscope (SEM)
Transmission
Electron
Microscope (TEM)
TEM is electron
illuminated. This
SEM use electron illumination. The image
A dissection microscope is light
This microscope uses a laser light. This
gives a 2-D view.
is seen in 3-D. It has high magnification
illuminated. The image that appears is light is used because of the wavelength.
Thin slices of
and high resolution. The specimen is
three dimensional. It is used for dissection Laser light scan across the specimen with
specimen are
coated in gold and the electrons bounce
to get a better look at the larger
the aid of scanning mirrors. Then image is
obtained. The
off to give you and exterior view of the
specimen. You cannot see individual cells then placed on a digital computer screen
electron beams pass
specimen. The pictures are in black and
because it has a low magnification.
for analyzing.
through this. It has
white.
high magnification
and high resolution.
$150 - $10,000
$100-$1500
$20,000-100,000
more than $50.000
more than $50,000
visible light
visible light
laser light
electrons
electrons
air
air
air
vacuum
vacuum
glass slides with dyed samples
Mounted on aluminum stubs and are
coated in gold
Thin films of
collodion or other
supporting material
on copper grids
one electrostatic
lens and a few
electromagnetic
lenses
glass slides
none
glass
glass
glass lenses with dichromatic mirrors
one electrostatic lens with a few
electromagnetic lenses
mechanical
mechanical
digital computer motorized focusing
mechanism
electrical
Electrical i.e. current
of the objective lens
coil is changed.
changing objectives
usually 1 objective
digitally enhanced
electrical
Electrical i.e.
changing current of
the projector lens
coil
Light Absorption
light scattering or light reflection
laser light with dicromatic mirror
concentrated at pinhole
electron scattering
Electron scattering
Fura-2 Calcium
sensitive
flourochrome
Green: High Ca
Blue: Low Ca
Why are Calcium concentration moving?
Lysosomes and
Mitochondria
Green: Mitochondria
stained with
Mitotracker Green
Red: Lysosomes stained
with LysoTracker Red
Immunofluorescence
Evans Blue Stain (non-specific red)
Yellow green fluorescing antibody for GLUT 2 transporters
Deconvolution Fluorescence
DNA: Blue
Microtubules: Green
Actin microfilaments: Red
Plasma Membrane
•A microscopic membrane of lipids and proteins that forms the external boundary of the cytoplasm of a cell or encloses a
vacuole, and that regulates the passage of molecules in and out of the cytoplasm
Mitochondria
•An organelle found in large numbers in most cells, in which the biochemical processes of respiration and energy
production occur. It has a double membrane, the inner layer being folded inward to form layers (cristae)
Lysosomes
•An organelle in the cytoplasm of eukaryotic cells containing degradative enzymes enclosed in a membrane
Nuclear envelope
•Double membrane forming the surface boundary of a eukaryotic nucleus; consists of outer and inner membranes
perforated by nuclear pores.
Nucleolus
•Subcompartment within the nucleus that is involved primarily in making ribosome components.
SER
•synthesize lipids, steroids and morphine, metabolize carbohydrates and steroids (but not lipids), and regulate calcium
concentration, drug metabolism, and attachment of receptors on cell membrane proteins
RER
•The surface of the rough endoplasmic reticulum (RER) is studded with protein-manufacturing ribosome's giving it a
"rough" appearance
Golgi Complex
•An organelle in eukaryotic cells consisting of stacks of flat membranous sacs that modify, store, and route products of the
endoplasmic reticulum.
Secretory Vesicles
•A membrane-bound organelle in which molecules destined for export are stored prior to their release, or exocytosis
Peroxisomes
•A small organelle that is present in the cytoplasm of many cells and that contains the reducing enzyme catalase and usually
some oxidases
Cytoskeletal Fibers
•The cytoskeleton is unique to eukaryotic cells. It is a dynamic three-dimensional structure that fills the cytoplasm. This
structure acts as both muscle and skeleton, for movement and stability. The long fibers of the cytoskeleton are polymers of
subunits. The primary types of fibers comprising the cytoskeleton are microfilaments, microtubules, and intermediate
filaments.
Microvilli
•microscopic cellular membrane protrusions that increase the surface area of cells, and are involved in a wide variety of
functions, including absorption, secretion, cellular adhesion, and mechanotransduction.
Cell Wall
•usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and
provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell
wall is to act as a pressure vessel, preventing over-expansion when water enters the cell. They are found in plants, bacteria,
fungi, algae, and some archaea. Animals and protozoa do not have cell walls.
Vacuole
•membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells.Vacuoles
are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including
enzymes in solution, though in certain cases they may contain solids which have been engulfed
Chloroplast
•found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to
conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called
photosynthesis.
Electron
Micrographs
Cultured mammalian cells
treated with gold particles
(black areas)
EE: Early Endosome
LE: Late Endosome
AV: autophagosomes
Rat Liver
P: Peroxisomes
M: Secondary Lysosome
containing mitochondria
fragments
Type of
Cells/Organ?
Profoundness of
Glycogen in this
cell/location?
Random act of
placement?
Cell Culture
http://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics.html