fluorescence microscopy fluorescent molecule = fluorochrome - absorbs light of specific wavelength -...

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)