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 Frequency of Event WAVELENGTH EMISSION SPECTRA Fluorochrome DAPI FITC Rhodamine

Fluorescence Microscopy Components: Light source Excitation filter Emission filter Dichroic mirror -reflects short -passes long SPECIMEN EYE PIECE

Texas Red- Acetylated TUBULIN FITC  ACTIN 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.

Fluorochrome PHOTOBLEACHING

(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

CONFOCAL MICROSCOPY Light source: laser illumination with coherent light

CONFOCAL MICROSCOPY Collects light from one plane of the sample at a time Excludes out of focus light scatter

REGULAR FLUORESCENCE CONFOCAL MICRSCOPE MICROSCOPE

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

Scanning Electron Microscopy (SEM) Surface structure Sectioning not required Metal coating of specimen Electron scattering Primary electrons Secondary electrons Detector

Pollen Ant Head

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 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 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

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. 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 com.bioquantusers.org/products.php?page=ls&content=gall ery&sub=feulgen

Goblet cells PAS stain Intestinal Villus

Carbohydrate-rich Basal Laminae stain with PAS stain

Staining Techniques Localization (staining) of an enzyme AB + T AT + B ENZYME generate visible product provide substrate

Staining Techniques AB + T AT + B Acetylcholinesterase- neuromuscular junction ACETYL CHOLINESTERASE 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)