BME 210 Lecture 4 Fluorophores - 4. Fluorophores Cells are...

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Unformatted text preview: 4. Fluorophores Cells are not fluorescent above 450 nm Exogenous fluorophores are used for imaging of: Proteins DNA, genes, RNA Intracellular organelles Lipids Ions of Ca, K, H, Na, Cl, Mg Transmembrane potential Fluorophore types 1) Organic molecules 2) Bioconjugates (fluorophore-biomolecule conjugates) 3) Fluorescent proteins 4) Quantum dots Important fluorophore properties 1. Optical spectra, wavelengths for peak absorption and emission 2. Fluorescence intensity: F ~ Q - extinction coefficient (measure of the ability to absorb light) Q- quantum yield (the ratio of the number of photons emitted to the number of photons absorbed, between 0 and 1) 3. Membrane permeability 4. Photostability (photobleaching) 5. Toxicity, phototoxicity Molar extinction coefficient: I 1 = I o e-lc (Beer-Lambert law) I 1 = I o 10- lc = -log(I 1 /I o )/(lc) = cm-1 M-1 Generic organic fluorophores (dyes) Fluorescein peak ex=495, em=515 peak ex=554, em=580 Rhodamine B =70,000/(M*cm) Q = 0.8 Q = 0.3 Alexa Fluor Set Alexa Fluor dyes - derivatives of fluorescein, rhodamine and cyanine dyes More photostable, less pH sensitive than the original dyes Hoechst 33342 346 460 DAPI 359 461 POPO-1 434 456 YOYO-1 491 509 Thiazole Orange 509 525 TOTO-1 514 533 Ethidium Bromide 526 604 Propidium Iodide 536 620 7-Aminoactinomycin D (7AAD)...
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BME 210 Lecture 4 Fluorophores - 4. Fluorophores Cells are...

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