1 / 12

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

rico
Télécharger la présentation

Fluoresecnce Correlation Microscopy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related