Transcript Lecture 4 Techniques III

Fluorescence Microscopy
Fluorescent molecule = fluorochrome
- absorbs light of specific wavelength
- when excited by absorption, the
fluorochrome emits light of longer
wavelength
Every fluorochrome has an absorption
and emission spectra.
Fluorescence Microscopy
Fluorochrome
Structurally unstable when excited
Fluorescence Microscopy
EXCITATION SPECTRA
EMISSION SPECTRA
WAVELENGTH
Fluorochrome
DAPI
FITC
Rhodamine
Fluorescence Microscopy
SPECIMEN
Components:
Light source
Excitation filter
Emission filter
Dichroic mirror
-reflects short 
-passes long 
EYE PIECE
Frog Neuromuscular Junction
Texas RedAcetylated
TUBULIN
FITC
ACTIN
By Stephanie Moeckel-Cole
Flurochromes can be used in combination to
mark different structures and/or
molecules.
“Multicolor "DiOlistic" labeling of the nervous
system using lipophilic dye combinations.” Gan
WB, Grutzendler J, Wong WT, Wong RO, Lichtman JW.
Labeled
neurons in
brain with
different
combination of
fluorochromes.
Fluorchromes:
DiO
DiI
DiD
FLUORESCENCE MICROSCOPY
PROBLEM: Photobleaching (fading)
Photobeaching: Fluorochrome loses ability
to fluoresce, absorb and emit light, due to
damage or covalent modification.
http://microscopyu.com/articles/fluorescence/fluorescenceintro.html
Fluorochrome
PHOTOBLEACHING
(a-f) Images collected at 2 minute intervals.
http://microscopyu.com/articles/fluorescence/fluorescenceintro.html
Quantum Dots : semiconductor nanoparticles, such as
cadmium selenide, that emitted light after light excitation.
Advantages: brighter, no photobleaching, broad excitation 
Disadvantages: potential toxicity for in vivo imaging
Alivisatos et al.; Quantum dots as cellular probes.; Annu
Rev Biomed Eng. 2005;7:55-76.
FLUORESCENCE MICROSCOPY
PROBLEM: Image degradation (blurring effect)
due to light scattering
http://www.microscopyu.com/articles/confocal/confocalintrobasics.html
CONFOCAL MICROSCOPY
Light source: laser
illumination with coherent light
http://hyperphysics.phy-astr.gsu.edu/hbase/optmod/qualig.html#c4
CONFOCAL MICROSCOPY
Collects light from one plane of the sample at a time
Excludes out of focus light scatter
REGULAR FLUORESCENCE
MICROSCOPE
CONFOCAL MICRSCOPE
CONFOCAL MICROSCOPY
Collect series of images from different focal planes
Can assemble the image series to yield a 3-d image
Transmission Electron
Microscopy (TEM)
Very thin section
Beam of electrons
(=0.005 nm)
Electromagnetic lenses
Stain with metals
Stain: electron dense: dark
Unstained: light
Nerve- osmium=myelin
http://www.utsa.edu/tsi/assign/histo/Images/histst1.gif
www.itg.uiuc.edu/ms/techniques/
Scanning Electron Microscopy (SEM)
Surface structure
Sectioning not required
Metal coating of specimen
Electron scattering
Primary electrons
Secondary electrons
Detector
http://www.chm.bris.ac.uk/pt/diamond/jamespthesis/chapter2_files/image002.gif
Ant Head
Pollen
FREEZE FRACTURE
Purpose: to analyze the distribution and
density of integral membrane proteins in
cell membranes
Freeze a fragment of tissue
Fracture using a sharp metal blade
-fracture plane passes through lipid
bilayers of a cell membrane
Observe with SEM
FREEZE FRACTURE
Cell Membrane: Lipid Bilayer
EXTRACELLULAR SPACE
CYTOPLASM
FRACTURE
EXTRACELLULAR SPACE
CYTOPLASM
FREEZE FRACTURE
EXTRACELLULAR SPACE
Cell Membrane
CYTOPLASM
P face: inner face of inner membrane.
EXTRACELLULAR SPACE
CYTOPLASM
E face: inner face of the outer membrane.
Intestinal Epithelium
Microvilli
Zonula adherens
Neuromuscular Junction
Synaptic site:
Active Zone: release
site of synaptic vesicles
Heuser and Reese
STAINING
Histochemistry or Cytochemistry: dyes
bind to certain types of molecules
Charged dyes bind to molecules of
opposite charge
Charged dyes bind to molecules of
opposite charge
Acidic Dye
---> dye
Eosin
Extracellular fibers, cytoplasmic filaments, and others
Basic Dye
---> dye +
Toluidine Blue
Alcian Blue
Cresyl Violet
Hematoxylin
Nuclei acids, glycosaminoglycans, ribosomes
Hematoxylin and Eosin
Intestine
Staining Techniques
There are many dyes.
http://medinfo.ufl.edu/~dental/denhisto/stains.html
Examples:
Sudan black
-Lipids
Myelinated axons- blue
ihcworld.com/imagegallery/displayimage.php?al...
Weigert Stain
-Reticular fibers
Staining Techniques
Histochemical Stains: involve chemical reactions
Feulgen reaction
-DNA
http://bioquantcom.bioquantusers.org/products.php?page=ls&content=gall
ery&sub=feulgen
Periodic Acid Shiff (PAS)
-neutral and acidic polysaccharides
- glycogen, mucous, basal laminae
Intestinal Villus
Goblet cells
PAS stain
Carbohydrate-rich Basal Laminae stain with PAS stain
Staining Techniques
Localization (staining) of an enzyme
AB + T
ENZYME
AT + B
provide substrate
generate visible product
Staining Techniques
AB + T
ACETYL
CHOLINESTERASE
AT + B
Acetylcholinesterase- neuromuscular junction
Other stains for ATPases, alkaline phosphatases, and others
IMMUNOCYTOCHEMISTRY
A technique to localize specific molecules in an
organ, tissue or cell.
First, a bit of immunology……….
An organism creates antibodies to foreign
molecules, ANTIGENS.
An antigen may have different regions,
EPITOPES, that are recognized as foreign by
an organism.
Polyclonal antibodies
-A collection of distinct types of antibody molecules
that recognize the same antigen (antibodies A + B + C)
but often several different epitopes
Monoclonal antibodies
-A single type of antibody molecule that recognizes only
one epitope on an antigen (antibody A OR B OR C)