Theory, Instrumentation and Vital Applications
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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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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

  • < 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


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


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


Mathematical Treatments

  • 2D Model Equation

  • 3D Model Equation

  • Active Transport


Potentially Accessible Vital Phenomena

  • Include:

  • Mobility and transport

    • Local absolute concentrations

    • Association / Dissociation Enzyme product formation

    • Photophysical phenomena

    • Compartmental environments


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??


Applications - Cellular Hormone Binding

Scatchard Analysis of FCS Data

Two distinct binding processes

2 X 1010 M-1

1 X 109 M-1

Specificity


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


FCS Curves

Applications

Fluid ordered SM

Fluid-disordered DOPC


The End


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