Download - FCS Basic Ideology
Expert Supervision By:Dr. C.K.NandiAssociate professorSchool Of Basic SciencesI.I.T Mandi
Presentation By:Kush KaushikV16016M.Sc ChemistryI.I.T Mandi
Fluorescence Correlation Spectroscopy (FCS) On Dynamics of Protein Corona
Why FCS?• In 1972 Watt Webb’s laboratory at Cornell put fluorescence microscopy to new use• Studied reaction kinetics• Characterising molecular Interactions in Vitro and Vivo• Ethidium bromide binding to DNA
• Individually don’t fluoresce but together glow under UVDr. Watt Webb
Source:Cornell.edu
What Happens When Light Strikes Something?• Absorption• Scattering• Emission
(Dynamic Light Scattering)
(Fluoroscence Correlation Spectroscopy)
Source- Self
Triplet state
Fluorescence
S1
S2
S3
S4
T1T2
FCS- Typical setup
Source: olympus.com
Particle Number Fluctuation
FCS – counting singlemolecules
Diffusion induces fluctuations of the number of molecules
N = 3 N = 2N = 4<N> = 3
I(t)
<I>This results in fluctuations of the fluorescence signal
t
Creating the Autocorrelation Function
“Copy” signal
Photon Burst
dI(t)
dI(t+t)
t=0 t=tD t=inf
FCS – autocorrelation analysis
dI(t)
0 t
G(t)
dI t dI t
t
2
I t
GO t
log t
FCS – autocorrelation analysis
dI(t)
0 t
G(t)
dI t dI t
t
2
I t
GO t
log t
FCS – autocorrelation analysis
dI(t)
0 t
G(t)
dI t dI t
t
2
I t
GO t
log t
FCS – autocorrelation analysis
dI(t)
0 t
G(t)
dI t dI t
t
2
I t
GO t
log t
FCS – autocorrelation analysis
G(t)
1/N 1/c
log t
tcorr 1/D
Fitting the autocorrelation function to appropriate model functions results in• properties of the diffusion process• the concentrationof several species with different hydrodynamic properties
The Effects of Particle Size on theAutocorrelation Curve
300 um2/s90 um2/s71 um2/s
Diffusion Constants
Fast Diffusion
Slow Diffusion
0.25
0.20
0.15
0.10
0.05
0.00G
(t)
10-7 10-6 10-5 10-4 10-3
Time (s)
Dk T
6r
Stokes-Einstein Equation:
)3/1(^/ MkD
and
Monomer --> Dimer Only a change in D by a factor of 21/3, or 1.26
The Effects of Particle Concentration on theAutocorrelation Curve
<N> = 4
<N> = 2
0.5
0.4
0.3
0.2
0.1
0.0
G(t
)
10-7 10-6 10-5 10-4 10-3
Time (s)
NNVarianceG 1)0( 2
2)(
)()()(
tF
tFtFG
tddt
Source- Czech Technical University
0.25
0.20
0.15
0.10
0.05
0.00
G(t
)
10-7 10-6 10-5 10-4 10-3
Time (s)
Autocorrelation of EGFP & Adenylate Kinase -EGFP
*EGFP is a Green Fluoresceine Protein
Source:Enrico gratton Lectures
Anti-Digoxin Antibody (IgG)Binding to Digoxin-Fluorescein
120
100
80
60
40
20
0
Frac
tion
Lig
and
Boun
d
10-10
10-9
10-8
10-7
10-6
[Antibody]free (M)
S. Tetin, K. Swift, & , E, Matayoshi , 2003
Protein Corona on Nano Particle
Spherical nanoparticle Protein MoleculesProtein Bounded on NP surface
Nature Nanotechnology8,701–702 (2013)
Nano particle
Plasma Adsorbed on NPSurface
Conclusion
• With The help of FCS, we can study the Interaction of Nano Particle with the protein.also the width of the protein absorbed on the surface.
• FCS is very userful in the study of molecular Binding
• Reaction kinetics can be studied
• But for getting FCS, the molecule must be fluorescence active
ACKNOWLEDGEMENT
we want to First thank the IIT Mandi to give us world Class Facilities, and also our Guide Dr. C.K Nandi Sir.We also want to acknowledge Dr. Nandi's Research Scholars Mr. Navneet Verma and Mr. Syamantak Khan for making us understasnd the research papers and also I want to acknowledge my Friend Shivendra Singh and Richa Garg for Helping in making the presentation
#####Thank You#####
Regards,Kush Kaushik
1
Expert Supervision By:Dr. C.K.NandiAssociate professorSchool Of Basic SciencesI.I.T Mandi
Presentation By:Kush KaushikV16016M.Sc ChemistryI.I.T Mandi
Fluorescence Correlation Spectroscopy (FCS) On Protein Corona
What is Fluorescence
• Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation.
Fluctuations Carry the Information• Measured intensity fluctuations reflects
(mobile fraction only) • Number of particles
• concentration• Diffusion of particles
• interaction• Brightness
• Oligomerization
• A particle that transits the confocal volume will generate groups of pulses.
• The correlation function calculates the mean duration time t of these groups.
• The variance/histogram of the signal yields information about oligomeric state
<I(t)>
dI(t)
dI(t)
Slow and Fast Fluctuation-Effect
Why Confocal Microscope
The Effects of Particle Size on theAutocorrelation Curve
300 um2/s90 um2/s71 um2/s
Diffusion Constants
Fast Diffusion
Slow Diffusion
0.25
0.20
0.15
0.10
0.05
0.00G
(t)
10-7 10-6 10-5 10-4 10-3
Time (s)
Dk T
6r
Stokes-Einstein Equation:
3rVolumeMW
and
Monomer --> Dimer Only a change in D by a factor of 21/3, or 1.26
Time (s)
G(t)
EGFPsolution
EGFPcell
EGFP-AKb in the cytosol
EGFP-AK in the cytosol
Normalized autocorrelation curve of EGFP in solution (•), EGFP in the cell (• ), AK1-EGFP in the cell(•), AK1b-EGFP in the cytoplasm of the cell(•).
Autocorrelation of EGFP & Adenylate Kinase -EGFP
Why Confocal Volume
• extraction of information of single particle.• as No. of Particle Increases, The observation of single molecule is difficult.
• Therefore, We need less no. of particles, which can be done by
-> Decreasing the Observation Volume.-> High Dilution.
Why Confocal Microscope
• We have two options now:->Decrease the Cuvette Size(to Fermi Litre).->Decrease the Concentration to 10^-24M.
• Both are impractical in Real world.• Confocal Microscope allows us to get such a impractical volume
i.e to 10-15L
Particle Number Fluctuation
• Particle moves with Different speeds• Speed is Described by the Difffusion Coefficent,
measured by the FCS
• Detects properties by the Fluctuations of Particle
• Particle number at time t. N(t) = <N> + ƌN
• Particle with high Diffusion coefficient shows fast fluctuations.
Why Confocal Microscope
• For a Typical Fluorimetre Cuvette(V=100uL, Solution Diluted to 100nM
• if <N> = 6 x 10^12 Particles
• Relative Fluctuations(ƌNSingle/N) = .00004%
• Which is very small to give valid Results
Why Confocal Microscope
Why Confocal MicroscopeConfocal Volume
120
100
80
60
40
20
0
Frac
tion
Lig
and
Boun
d 10
-1010
-910
-810
-710
-6
[Antibody]free (M)
Digoxin-Fl•IgG(99% bound)
Digoxin-Fl
Digoxin-Fl•IgG (50% Bound)
Autocorrelation curves:
Anti-Digoxin Antibody (IgG)Binding to Digoxin-Fluorescein
Binding titration from the autocorrelation analyses:
triplet state
Fb mSfreeKd S free
c
Kd=12 nM
S. Tetin, K. Swift, & , E, Matayoshi , 2003
FCCS – fluorescence cross correlation spectroscopy
Extended concept:• labeling of potential binding partners with spectrally different fluorophores• looking for correlations between the corresponding signals
I(t) no correlationG(t)
t log t
I(t) correlationG(t)
kaskdis
t log t
FCCS – model application
kas
+
kdis
G(t)
log t
FCCS – model application
kas
+
kdis
G(t)
log t
FCCS – model application
kas
+
kdis
G(t)
log t