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Fluoresecnce Correlation Microscopy

Theory, Instrumentation and Vital Applications. Fluoresecnce Correlation Microscopy. Overview . Problems with fluorescence methodology Variation of relaxational methods Monitors minute intrinsic changes in fluorescence. Overview. Fluorescence Correlation Spectroscopy

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Fluoresecnce Correlation Microscopy

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  1. Theory, Instrumentation and Vital Applications Fluoresecnce Correlation Microscopy

  2. Overview • Problems with fluorescence methodology • Variation of relaxational methods • Monitors minute intrinsic changes in fluorescence

  3. Overview Fluorescence Correlation Spectroscopy • < 1 fL focal volume • Measures the residence time and the changes in fluorescence intensity that occur while the molecule is localized within the focal volume • High spatial and temporal resolution at low [fluorophore] Image courtesy of Schwille, Haustein Book Chapter, FCS

  4. FCS Instrumentation • First applied to solution studies • Later adapted to fluorescence microscopy • Later combined with confocal imaging • Can be adapted to common epifluorescence microscopes!!! • Requires laser source, hardware correlator and pinhole barrier between emitted radiation and a APD Pinhole 30 nm diameter Schwille and Haustein, Fluorescence Correlation Spectroscopy

  5. Theoretical Brownian diffusion behaviour D = kbT / 6R Correlation time D = o2 / 4D Therefore the average dwell time for a freely diffusing molecule is about 170 sec

  6. Mathematical Treatments • 2D Model Equation • 3D Model Equation • Active Transport

  7. Potentially Accessible Vital Phenomena • Include: • Mobility and transport • Local absolute concentrations • Association / Dissociation Enzyme product formation • Photophysical phenomena • Compartmental environments

  8. Applications - Cellular Hormone Binding Membrane-bound Rh-Insulin Free Rh-Insulin • Insulin • Receptor levels diagnostic for Type II diabetes • Typically done by radioligand assays • drawbacks • By FCS using rhodamine labeled insulin • Receptor aggregation or multiple sites??

  9. Applications - Cellular Hormone Binding Scatchard Analysis of FCS Data Two distinct binding processes 2 X 1010 M-1 1 X 109 M-1 Specificity

  10. Applications – Lipid Dynamics DiI-C18 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate Preferentially localizes to regions containing saturated, long-chain phospholipids Excluded from sphingomyelin GM1 ganglioside sphingomyelin or glycosphingolipid Raft marker that localizes to sphingomylein rich regions Binds cholera toxin B subunit with high affinity Cholesterol also preferentially localizes to sphingomyelin rich regions in ‘raft structures’ DOPC / Sphingomyelin / Cholesterol

  11. FCS Curves Applications Fluid ordered SM Fluid-disordered DOPC

  12. The End

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