fluorescence microscopy fluorescent molecule = fluorochrome
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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 SPECTRAF
r equ
enc
y o
f Eve
nt
Fr e
que
ncy
of E
vent
WAVELENGTH
EMISSION SPECTRA Fluorochrome DAPI FITC Rhodamine
Fluorescence Microscopy
Components:Light sourceExcitation filterEmission filter
Dichroic mirror-reflects short -passes long
SPECIMEN
EYE PIECE
Texas Red-Acetylated TUBULIN
FITCACTIN
Frog Neuromuscular Junction
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
http://microscopyu.com/articles/fluorescence/fluorescenceintro.html
(a-f) Images collected at 2 minute intervals.
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 MICROSCOPYLight source: laser
illumination with coherent light
http://hyperphysics.phy-astr.gsu.edu/hbase/optmod/qualig.html#c4
CONFOCAL MICROSCOPYCollects light from one plane of the sample at a time
Excludes out of focus light scatter
REGULAR FLUORESCENCE CONFOCAL MICRSCOPE MICROSCOPE
CONFOCAL MICROSCOPYCollect 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 metalsStain: electron dense: darkUnstained: light
Nerve- osmium=myelinhttp://www.utsa.edu/tsi/assign/histo/Images/histst1.gif
www.itg.uiuc.edu/ms/techniques/
Scanning Electron Microscopy (SEM)
Surface structureSectioning not required
Metal coating of specimenElectron scatteringPrimary electronsSecondary electronsDetector
http://www.chm.bris.ac.uk/pt/diamond/jamespthesis/chapter2_files/image002.gif
Pollen
Ant Head
FREEZE FRACTUREPurpose: 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
CYTOPLASM
EXTRACELLULAR SPACE
Cell Membrane: Lipid Bilayer
CYTOPLASM
EXTRACELLULAR SPACE
FRACTURE
P face: inner face of inner membrane.
FREEZE FRACTURE
CYTOPLASM
EXTRACELLULAR SPACE
E face: inner face of the outer membrane.
CYTOPLASM
EXTRACELLULAR SPACE
Cell Membrane
Intestinal Epithelium
MicrovilliZonula 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 EosinIntestine
Staining Techniques
There are many dyes.
http://medinfo.ufl.edu/~dental/denhisto/stains.html
Examples:Sudan black
-Lipids
Weigert Stain-Reticular fibers
Myelinated axons- blue
ihcworld.com/imagegallery/displayimage.php?al...
Staining Techniques
Histochemical Stains: involve chemical reactions
Feulgen reaction -DNA
Periodic Acid Shiff (PAS)
-neutral and acidic
polysaccharides
- glycogen, mucous, basal
laminae
http://bioquant-com.bioquantusers.org/products.php?page=ls&content=gallery&sub=feulgen
Goblet cells PAS stainIntestinal Villus
Carbohydrate-rich Basal Laminae stain with PAS stain
Staining Techniques
Localization (staining) of an enzyme
AB + T AT + BENZYME
generate visible product
provide substrate
Staining Techniques
AB + T AT + B
Acetylcholinesterase- neuromuscular junction
ACETYLCHOLINESTERASE
Other stains for ATPases, alkaline phosphatases, and others
A technique to localize specific molecules in an organ, tissue or cell.
IMMUNOCYTOCHEMISTRY
An organism creates antibodies to foreign molecules, ANTIGENS.
An antigen may have different regions, EPITOPES, that are recognized as foreign
by an organism.
First, a bit of immunology……….
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)