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Fluoresecnce Correlation Microscopy
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Transcript of Fluoresecnce Correlation Microscopy
Fluoresecnce
Correlation
Microscopy
Theory, Instrumentation
and Vital Applications
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
Schwille and Haustein, Fluorescence Correlation SpectroscopyPinhole 30 nm diameter
Theoretical Brownian diffusion behaviour
D = kbT / 6R
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 concentrationsAssociation / Dissociation Enzyme product formation Photophysical phenomenaCompartmental environments
Applications - Cellular Hormone Binding
• 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??
Free Rh-Insulin Membrane-bound Rh-Insulin
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
ApplicationsFluid-disordered DOPC Fluid ordered SM
FCS Curves
The End