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Spectroscopic

Microscopy

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Limitation of light microscopy

5 mm

limit ofresolution

lblue

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Confocal Microscopy

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Absorption Spectra of Fluors CommonlyConjugated to Secondary Antibodies

Fluorochrome Absorption Emission

Cascade Blue 400 420Fluorescein 494 518Rhodamine 570 590Texas Red 595 615Cy5 650 670

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Colocalization of insulin and calcitonin receptor-like receptor

Insulin-Cy5 CRLR-FITC

Glucagon-Rhodamine

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Fluorescence Resonance Energy Transfer

FRET is the non-radioactive transfer of photon energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when both are located within close proximity (1-10nm).Using FRET one can resolve the realtive proximity of molecules beyond the optical limit of a light microscope to reveal (1) molecular interactions between two protein partners,(2) structural changes within one molecule (eg. enzymatic activity or DNA/RNA conformation),(3) ion concentrations using special FRET-tools like the CFP-YFP cameleon

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No FRETSignal

FRETSignal

CFP is excited by light and emits lightCFP is more than 10nm from YFPYFP is not excited and does not emit light

CFP is excited by light and emits lightCFP is in close proximity to YFPYFP emits light

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FLIM (Fluorescence Lifetime Imaging Microscopy)

• measures the lifetime of the excited state (delay between excitation and emission)

• every fluorophore has a unique natural lifetime• lifetime can be changed by the environment, such

as: Ion concentration Oxygen concentration pH Protein-protein interactions

∆t=lifetime∆t=lifetime

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FRAP (Fluorescence Recovery After Photobleaching)

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