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

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

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.


• 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.


• 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 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