Powerpoint Templates Page 1 Powerpoint Templates Spectroscopic Microscopy.

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Presentation transcript:

Powerpoint Templates Page 1 Powerpoint Templates Spectroscopic Microscopy

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Powerpoint Templates Page 3 Limitation of light microscopy 5  m limit of resolution blue

Powerpoint Templates Page 4 Confocal Microscopy

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Powerpoint Templates Page 6 Absorption Spectra of Fluors Commonly Conjugated to Secondary Antibodies Fluorochrome AbsorptionEmission Cascade Blue Fluorescein Rhodamine Texas Red Cy

Powerpoint Templates Page 7 Colocalization of insulin and calcitonin receptor-like receptor Insulin-Cy5 CRLR-FITC Glucagon-Rhodamine

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Powerpoint Templates Page 10 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

Powerpoint Templates Page 11 No FRET Signal FRET Signal CFP is excited by light and emits light CFP is more than 10nm from YFP YFP is not excited and does not emit light CFP is excited by light and emits light CFP is in close proximity to YFP YFP emits light

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Powerpoint Templates Page 13 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

Powerpoint Templates Page 14 FRAP (Fluorescence Recovery After Photobleaching)

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