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