TwoPhoton Fluorescence Correlation Spectroscopy

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Two-Photon Fluorescence Correlation Spectroscopy

Zifu Wang Beckman Laser Institute Optical
Biology Center
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Introduction

• Fluorescence correlation spectroscopy (FCS) has
  recently experienced growing popularity in
  biochemical and biophysical applications due to
  significantly improved signal-to-background
  ratios. The parameters determinable by FCS in
  intracelluar environment include

• Fluorescent species (e.g., GFP, beads, dye
  molecules) diffusion coefficients
• Fluorescent molecules concentration
• Kinetic information of binding and aggregation
  processes

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What is Fluorescence Correlation Spectroscopy
(FCS)?

• Diffusion
• Fluorescent molecules diffusing in and out of the
  volume element result in fluctuations of the
  fluorescence intensity measured from that small
  volume element.

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What is FCS? (Cont.)
Intensity
Time t
(fluctuating fluorescence signal detected by
photon detector)
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2-Photon FCS Experimental Setup
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2-Photon FCS Experimental Setup
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What is FCS? (Cont.)
The normalized fluorescence fluctuation
correlation function is defined as
G(t)?dI(t)dI(tt)?/?I(t)? 2

FCS autocorrelation functions representing a low
molecular weight ligand (left curve, blue),
macromolecule-bound ligand (right curve, red) and
a 11 mixture of free and bound ligand (middle
curve, green).
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Principles of Fluorescence Correlation
Spectroscopy (FCS) (Cont.)

• Polymerization and Hybridization (binding)
• The processes of polymerization and binging could
  be detected though FCS measurements.

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What is FCS? (Cont.)
The measured autocorrelation function will be
curve-fitted with the following analytical form
for single diffusion species
GD(t)1g/N(1t/tD)(1t/w2tD)0.51GN(t)

• The analysis provides us the following
  information
• Number (N) of diffusing molecules in the known
  focal volume of laser beam, which directly
  relates to the concentration of the molecules.
• Diffusion constant given by
• D r02 /8 tD
• where r0 is the radius of the excitation volume.
  tD is the measured characteristic diffusion time
  for the molecules to cross the focal volume of
  the laser beam. w is a constant (0.2-0.3). g is
  also a known constant.

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Calibration of a FCS system
D360 mm2/s
r00.34 mm
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ECFP protein in living PTK2 cells
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FCS application in cell biology
DTub6 mm2/s DCFP20 mm2/s
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Molecular brightness
h ?F(t)? /N
Molecular brightness is a important parameter
obtained from FCS measurements. It is dependent
on the intrinsic properties of the fluorophore
and design of optics and not affect by
concentration. Due to the stable structure of
GFP, its emission properties is not strongly
affected by external environments. h can be used
to study the oligomerization state of tubulin in
vivo.
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FCS application to mitotic checkpoint study
D1.8 mm2/s
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FCS application to developmental biology
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FCS application to developmental biology
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Analytical model of active transportation
G(t)1/Nexp(-(t/tf)2 tf is the average
residence time for the molecules within the
excitation volume of the laser beam. tf
r0/(n1.414). n is velocity, r0 is the lateral
dimension of the volume (0.34 mm) in our system,
and N is the number of molecules in the volume.
n0.006031518 (mm/s) 21.7 mm/hr