Whole Cell + Tissue Techniques
To answer the questions:
What does an organelle do? What is it made of? What proteins does it contain?
What does this protein do? How does it do it? Where in the cell is it located?
Subcellular Fractionation (Disruptive technique)
- Break cells (homogenisation), differential or
density gradient centrifugation to separate
organelles, collect fractions and assay fractions
for enzymes, antigens etc.
Quantitative (% distribution)
Results representative (averaged over whole
population)
Needs lots of cells/tissue (106+ cells)
Cannot detect inter-cell/organelle variability
Artefacts = redistribution due to organelle breakage
- Examples of organelle densities
Lysosomes: 1.12 g.cm-3
Mitochondria: 1.18 g.cm-3
Peroxisomes: 1.23 g.cm-3
Microscopy (Non-disruptive technique)
,- Fix cells to stop protein distribution, permeabilise (fluorescence microscopy) or
embed and section (electron microscopy), label for antigens etc.
Few cells needed
Can detect inter-cell/organelle variability
Cells have to be examined one by one
Need a significant number of cells for proper
quantification
Light Microscopy
- Magnify cells up to 1000x and resolbe details
as small as 0.2µm
Limitation imposed by wavelength of light
a) Bright light must be focused onto specimen
by lenses in the condenser
b) Specimen must be carefully prepared to
allow light to pass through it
c) An appropriate set of lenses (objective and
eyepiece) must be arranged to focus an
image of the specimen in the eye
- Living cells or fixed samples (cut into sections
and mounted onto a slide) – can be stained
Fluorescence Microscopy
- Fluorescent dyes used for staining cells detected
with the aid of fluorescence microscope
- Similar to an ordinary light microscope except that
the illuminating light is passed through two sets of
filters
- First filters the light before it reaches specimen,
passing only the wavelengths that excite particular
fluorescent dye
- Second blocks out excitatory wavelengths and allows
emission wavelengths when dye fluoresces
- Dyed objects show up in bright colour on a dark background
Fluorescent Probes
, - Dyes absorb light at one wavelength and emit it at
another
- Some dyes bind specifically to particular molecules
in cells
- some can be coupled to antibody molecules which
then serve as highly specific and versatile staining
reagents that bind selectively to particular
macromolecules allowing distribution in cell to be
visualised
Confocal Microscopy
- Specialised type of fluorescence microscope
that builds up an image by scanning specimen
with a laser beam
- Beam is focused onto a single point at
specific depth in the specimen, pinhole
aperture in the detector allows only
fluorescence emitted from the same point to
be included in the image
- Scanning the beam across the specimen generates a sharp image of the plane of
focus = optical section
- Series of optical sections at different depths
allows a 3D image to be constructed
- Conventional fluorescence microscopy gives a
blurry image due to presence of fluorescent
structures above and below plane of focus
Confocal microscopy provides an optical section
showing individual cells clearly
Examples
3D reconstruction of cell morphology from confocal sections of carcinoma cells
following EGF stimulation: phalloidin staining for F-actin cytoskeleton (maximum
density reconstruction)
Rat retinal wholemount in which retinal ganglin cells infected with rAAV/GFP
Tubulogenesis by HGF-treated MDCK cells grown in 3D collagen (Li and Pendergast)
Porcine conjunctiva, rat gut
Immunofluorescence Technique
To answer the questions:
What does an organelle do? What is it made of? What proteins does it contain?
What does this protein do? How does it do it? Where in the cell is it located?
Subcellular Fractionation (Disruptive technique)
- Break cells (homogenisation), differential or
density gradient centrifugation to separate
organelles, collect fractions and assay fractions
for enzymes, antigens etc.
Quantitative (% distribution)
Results representative (averaged over whole
population)
Needs lots of cells/tissue (106+ cells)
Cannot detect inter-cell/organelle variability
Artefacts = redistribution due to organelle breakage
- Examples of organelle densities
Lysosomes: 1.12 g.cm-3
Mitochondria: 1.18 g.cm-3
Peroxisomes: 1.23 g.cm-3
Microscopy (Non-disruptive technique)
,- Fix cells to stop protein distribution, permeabilise (fluorescence microscopy) or
embed and section (electron microscopy), label for antigens etc.
Few cells needed
Can detect inter-cell/organelle variability
Cells have to be examined one by one
Need a significant number of cells for proper
quantification
Light Microscopy
- Magnify cells up to 1000x and resolbe details
as small as 0.2µm
Limitation imposed by wavelength of light
a) Bright light must be focused onto specimen
by lenses in the condenser
b) Specimen must be carefully prepared to
allow light to pass through it
c) An appropriate set of lenses (objective and
eyepiece) must be arranged to focus an
image of the specimen in the eye
- Living cells or fixed samples (cut into sections
and mounted onto a slide) – can be stained
Fluorescence Microscopy
- Fluorescent dyes used for staining cells detected
with the aid of fluorescence microscope
- Similar to an ordinary light microscope except that
the illuminating light is passed through two sets of
filters
- First filters the light before it reaches specimen,
passing only the wavelengths that excite particular
fluorescent dye
- Second blocks out excitatory wavelengths and allows
emission wavelengths when dye fluoresces
- Dyed objects show up in bright colour on a dark background
Fluorescent Probes
, - Dyes absorb light at one wavelength and emit it at
another
- Some dyes bind specifically to particular molecules
in cells
- some can be coupled to antibody molecules which
then serve as highly specific and versatile staining
reagents that bind selectively to particular
macromolecules allowing distribution in cell to be
visualised
Confocal Microscopy
- Specialised type of fluorescence microscope
that builds up an image by scanning specimen
with a laser beam
- Beam is focused onto a single point at
specific depth in the specimen, pinhole
aperture in the detector allows only
fluorescence emitted from the same point to
be included in the image
- Scanning the beam across the specimen generates a sharp image of the plane of
focus = optical section
- Series of optical sections at different depths
allows a 3D image to be constructed
- Conventional fluorescence microscopy gives a
blurry image due to presence of fluorescent
structures above and below plane of focus
Confocal microscopy provides an optical section
showing individual cells clearly
Examples
3D reconstruction of cell morphology from confocal sections of carcinoma cells
following EGF stimulation: phalloidin staining for F-actin cytoskeleton (maximum
density reconstruction)
Rat retinal wholemount in which retinal ganglin cells infected with rAAV/GFP
Tubulogenesis by HGF-treated MDCK cells grown in 3D collagen (Li and Pendergast)
Porcine conjunctiva, rat gut
Immunofluorescence Technique
