2.
Cell biology tools
1. Microscopy
• Microscopes in general
- Condensed light in the visible range passes through the
specimen and is then captured by an objective lens. The light
absorbed by the sample produces contrasts revealing
structural detail.
- Limit of resolution: minimal distance between two closely
positioned objects at which the two subject can be observed to
be distinct entities
d= =
(d= limit of resolution/resolving power, λ= the wavelength of
light used, n= refractive index, NA= the numerical aperture of
the lenses)
· light microscopes: NAmax,air=1.0, NAmax,oil=1.4, λ=～400 nm
theoretical limit of resolution = 0.1 μm
best resolving power = 0.2 μm (1000-folds of magnification)
· electronic microscope: λ= ～0.0004 nm, NA= 0.02
theoretical limit of resolution : 0.1 nm
actual limit of resolution 1-2 nm (250,000-folds of
magnification)
- General procedure of light microscopy
(1) Fixation
· glutaraldehyde or formaldehyde (cross-linking of adjacent
protein molecules)
· osmium tetroxide (stabilization of lipid membranes and
proteins, also staining of the surfaces of membranes very
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well)
(2) Embedding in paraffin
(3) Thin sectioning: a microtome
(4) removing the paraffin-embedding medium with xylene and
then rehydrate the samples before staining
(5) Staining
· silver salts: reticular fibers in the extracellular matrix
· periodic acid-Schiff (PAS) reagent: glycoproteins and
carbohydrate-rich macromolecules
· hematoxylin and eosin (H&E): through simple charge
interactions/much less specific manner/most common
eosin (acidic dye): reacts with positively charged
macromolecules (cytoplasm and most of the
extracellular filaments - pink)
hematoxylin & a mordant (basic dye): bind to
negatively charged cellular structures (DNA, RNA &
rER - blue)
• Fluorescence Microscopy
- a form of light microscope with a series of filters that permit
the excitation and visualization of the fluorescent labeling of
specific proteins
- identification of subcellular location of particular proteins
through specific probing
(immunocytochemistry/immunostaining)
- antibodies for immunostaining
· primary antibodies/secondary antibodies
· monoclonal antibodies/polyclonal antibodies
- labeling (tagging)
· immunolabeling: fluorescence coupling to antibodies for LM,
2
ferritin or nanogold particle coupling to antibodies for EM
· genetic tagging: green fluorescence protein (GFP) tagging,
tetra-cysteine (Cys-Cys-X-X-Cys-Cys) tagging with green
FlAsH or red ReAsH, myc-tagging (epitope tagging)
cf. FRET (fluorescence resonance energy transfer)
• Other types of microscopes
- phase-contrast microscope
- differential interference contrast (DIC) microscope : with a
series of prisms and polarizers
- polarizing microscope
- dark-field microscope
- confocal scanning microscope
• Transmission Electron Microscopy
- As the specimen is bombarded by the electron beam, some of
electrons are deflected by the sample, whereas some others
pass directly through the specimen. Those electrons that pass
through the sample form an image by being focused onto a
phosphorescent screen for viewing.
- General procedure of TEM
(1) chemical fixation
· glutaraldehyde or formaldehyde (cross-linking of adjacent
protein molecules)
· osmium tetroxide (stabilization of lipid membranes and
proteins, also staining of membrane surface)
(2) Embedding in plastics
(3) Ultra-thin sectioning: a microtome
(4) Staining
· uranyl acetate or lead citrate
3
· osmium tetroxide
· colloidal gold (immunostaining)
• Modified EM procedures
- Metal-shadowing electron microscopy
· for fixed, frozen, and dried specimens
· coating with heavy metal (e,g., platinum)/viewing with TEM
- Freeze-fracture electron microscopy
· for membrane morphology
· rapid freezing at -180℃/splitting with a knife blade/coating
with platinum-carbon/removing tissues/viewing with TEM
- Cryoelectron Microscopy
· to view molecular structure at a high level of magnification
· rapid freezing/deep etching/shadowing with platinum/
viewing with TEM or SEM
· or computer-based averaging techniques → tomographic 3D
images in a few nanometer range
- Scanning electron microscopy (SEM)
· coating with a thin film of heavy-metal ions (gold or
gold-palladium) and then scanning with a electron beam
· accurate presentation of the cell surface feature (5 nm
resolution)
- Atomic-force microscopy (AFM)
· for real-time micrographs of samples in buffers or living
cells
· comparable resolution with SEM
*
used for nanodisection and measurement of interactive force
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2. Cell culture
- cell culture media: mimic the normal physiologic environment,
isoosmotically corrected mixtures of amino acids, salts,
vitamins and other components (e.g., glucose) + fetal calf
serum (FCS)
- The serum provides
(1) extracellular matrix proteins
(2) transferrin
(3) growth factors (PDGF, EGF, ILGF, etc.)
- primary cultured cells
· become senescent after many doublings
- established cell lines
· escaped from senescence, becoming immortal
· from cancer cells (e.g., HeLa cells)
3. Flow Cytometry
- a method to count and sort individual cells based on size,
granularity, and fluorescence intensity
- a fluorescence-activated cell sorter (FACS)
· measuring the characteristics of a cell population
(cytometry)
· sorting subpopulations of cells (cell sorting)
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4. Cell fractionation
- cell lysis by homogenization : a Dounce homogenizer
- differential centrifugation
· low speed: unlysed cells and intact nuclei (800 g, 10 min)
· high speed: mitochondria and lysosomes (15,000 g, 10 min)
· very high speed: membranous structures (100,000 g, 60
min) ribosomes (200,000 g, 3 h)
· desk-top centrifuge (up to 3,000 rpm)/high-speed centrifuge
(up to 25,000 rpm)/ultra-centrifuge (up to 80,000
rpm/500,000 g)
- buoyant density centrifugation (density gradient centrifugation)
․ a gradient of sucrose or salt
․ rate-zonal centrifugation: a gradient of low density (5-20%)
․ equilibrium density gradient centrifugation: a gradient of
high density (20-70%): stop moving when ρparticle = ρsolution
cf. sedimentation coefficient: S (Svedberg value, 10-13 sec)
5. Column chromatography
- bioactive macromolecules
- gel filtration chromatography
· porous beads (gel or resins)
· by size
- ion-exchange chromatography
· positively or negatively charged resins
· by charge
- hydrophobic interaction chromatography
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· resins with hydrophobic side chains
· by hydrophobicity
- affinity chromatography
· resins with specific ligands
· by a specific interaction (substrate/enzyme or Ag/Ab)
· fast and homogeneous preparation
6. Electrophoresis
- SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel
electrophoresis)
· SDS: negative charges to all proteins
· β-mercaptoethanol: disruption of disulfide bonds
· The rate of protein migration depends only on the relative
molecular mass.
- Western blotting
- two dimensional gel electrophoresis
a. Isoelectric focusing
b. SDS-PAGE
- Agarose gel electrophoresis
7. Other tools for cell biology
- microelectrodes
- patch clamping
- microinjection
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- mass spectrometry
· bombarding proteins with electrons → breaking into
charged fragments → separating according to their
mass-to-charge ratio in an electric or magnetic field
· identification of proteins and their post-translational
modifications
· localization of proteins to the various organelles, and
determination of the interactions between different proteins
and membrane lipids
- microarray
· 2D array on a solid substrate (usually a glass slide) that
assays large amounts of biological material using
high-throughput screening miniaturized, multiplexed and
parallel processing and detection methods
· types: DNA microarrays (cDNA microarrays, oligonucleotide
microarrays, etc.), protein microarrays, tissue microarrays,
cellular microarrays, etc.
