7.1 Fluorochromes Fluorescence microscopy differentiates between two kinds of fluorochromes:

1. 7. Fluorescence microscopy • 7.1 Fluorochromes • Fluorescence microscopy differentiates between two kinds of fluorochromes: • Primary fluorescence (autofluorescence) • Secondary fluorescence (fluorochromation) • Fluorescence dyes • Immunofluorescence (using Antibodies) • Molecular tags (SNAP Tag, ...) • Fluorescent Proteins • Applications of fluorochromes • Identification of otherwise visible structures • Localization and identification of otherwise invisible structures • Monitoring of physiological processes • Specific detection of a protein • Using Photophysical properties of dyes (e.g. switching) for superresolution

2. 7. Fluorescence microscopy 7.1 Primary fluorescence (autofluorescence) • Most samples fluoresce when excited with short-wave light • Fluorescence very often occurs for systems containing many conjugated double bonds: • e.g. chlorophyll exhibits dark red fluorescence when excited by blue or red light Porphyrin ring – central unit in Chlorophyll Moss reeds – green excitation

3. 7. Fluorescence microscopy http://en.wikipedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG

4. 7. Fluorescence microscopy 7.1 Primary fluorescence (autofluorescence) • Further examples: • Riboflavine (550nm) • NAD(P)H (460nm, 400ps) • Elastin und Collagen (305-450nm) • Retinol (500nm) • Cuticula (blue) • Lignin (> 590nm) • DNA (Ex @320nm, 390nm) • Aminoacids: • Tryptophane (348nm, 2.6ns) • Tyrosin (303nm, 3.6ns, weak) • Phenylalanine (282nm weak) • Resins, Oils Eucalyptus leaf section – UV excitation http://en.wikipedia.org/wiki/Autofluorescence Nematode living sample – UV excitation

5. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation) • Staining (labeling) specific structures with fluorescent labels (dyes): fluorochromation • Small dye concentrations are sufficient due to high fluorescence contrast  fluorescence labels are superior than bright field dyes • Single molecule sensitivity • Fluorescence labels must selectively bind to structures or selectively accumulate in specific compartments • e.g. DAPI (= 4',6-diamidino-2-phenylindole) to label DNA (cell nuclei) Fluorescence image of Endothelium cells. Microtubili are labeld in green, while actin filaments are labeled red. DNA within cell nuclei are stained with DAPI. DAPI: lexc = 358 nmlem = 461 nm some dyes unquench upon binding

6. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation): Immunofluorescence • Immunofluorescence (antibody staining) is the labeling of specific proteins with an antibody which is visualized by a dye • Antibodies are proteins which are used by the immune system to identify and neutralize foreign substances (antigens) • Antibodies are made of two large heavy chains (~440 amino acids) and two small light chains(~220 amino acids) • C-region is similar for all antibodies, while the V-regionis extremely variable and forms the specific binding sitefor the antigen i.e. every antibody can recognize andbind two antigens • Specificity: antibody-antigen reaction. The part of the antigen (protein) recognized by an antibody is called an epitope. Highly specific interaction, called induced fit, allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism

7. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation): Immunofluorescence • Different groups of antibodies exist: • Polyclonal antibodiesThey are a mixture of antibodies secreted against a specific antigen, each recognizing a different epitope i.e. bind to different areas of the protein. The protein (e.g. tubulin) for which a special antibody should be generated is injected into a suitable mammal (mostly rats, mice or goats).Antibodies against the protein are produced by the mammalian immune response and can be isolated from the blood serum • Monoclonal antibodiesare all identical and bind to the same epitope • Synthetic antibodiesare monoclonal antibodies which are produced in-vitro i.e. via microorganism • Other systemsscFv (M. Bruchez): single chain variable region anitbodiesnanobodies (H. Leohardt): small (from Camelidae) and not degrated quickly inside a cell

8. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation): Immunofluorescence • Direct immunofluorescence • For the direct or primary labeling the specific antibody for the investigated protein is labeled with the fluorochrome • The labeled antibodies are brought onto the sample and only bind specifically to the wanted protein (antigene = ligand); non bound antibodies are washed out • Detection of the bound antibodies via the attached fluorochrome  Localization of the wanted protein An interphase female human fibroblast cell. Arrow points to the corresponding X chromosome (right). Labeling of a DNA-associated histone protein

9. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation): Immunofluorescence • Indirect immunofluorescence • Two sets of antibodies; Primary antibody detects antigen A subsequent, secondary (indirect), dye-coupled antibody recognizes the primary antibody. • Signal amplification (several secondaries bind to one primary) • Color palette separating staining from target • Example: • 1. Antibody / Rat – Anti TubulinAntibody against tubulin generated in a rat • 2. Antibody / Goat – Anti Ratfluorescently labeled antibody against all rat antibodies (generated in a goat) • Negative test: Primary antibody is left out in order to test if the fluorescence labeled secondary antibody binds unspecifically to the sample

10. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation): Immunofluorescence Indirect immunofluorescence • GPCR transfected HEK cells: • Double staining: • For an identification of single cells the dye Hoechst 33342 was employed (cell nuclei: blue); • Cell bound primary mouse anti-GPCR antibodies were detected by secondary goat anti-mouse Ig(H+L) antibodies labeled with Alexa Fluor 488 (GPCR-protein: green). • The fluorescence labeled secondary antibodies can be employed for all antibodies produced within one animal e.g. goat serum against rats reacts with all primary antibodies produced in rats

11. 7. Fluorescence microscopy 7.1 Secondary fluorescence (fluorochromation) Dye artefacts • Bleaching: Fluorescence dye is destroyed by irradiation with light • Quenching: Fluorescence can be quenched (reduced) for large dye concentrations • Cross-Talk: Cross-Excitation: Simultaneous excitation of two dyes if their excitation wavelengths are too close to each other Bleed through: In case the emission spectra overlap too much both dyes will be detected but to different amounts Can be compensated by calibration and an inverse matrix technique "Spectral unmixing"