1. Expert Supervision By: Dr. C.K.Nandi Associate professor School Of Basic Sciences I.I.T Mandi Presentation By: Kush Kaushik V16016 M.Sc Chemistry I.I.T Mandi Fluorescence Correlation Spectroscopy (FCS) On Dynamics of Protein Corona 1

2. 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 UV Dr. Watt Webb Source:Cornell.edu 2

3. What Happens When Light Strikes Something? Absorption Scattering Emission (Dynamic Light Scattering) (Fluoroscence Correlation Spectroscopy) Source- Self 3

4. Triplet state Fluorescence S1 S2 S3 S4 T1 T2 4

5. FCS- Typical setup Source: olympus.com 5

6. Particle Number Fluctuation 6

7. FCS – counting singlemolecules Diffusion induces fluctuations of the number of molecules N = 3N = 2N = 4 = 3 I(t)I(t) This results in fluctuations of the fluorescence signal t 7

8. Creating the Autocorrelation Function “Copy” signal Photon Burst  I(t)  I(t+  )  =0 =D=D  =inf 8

9. FCS – autocorrelation analysis I(t)I(t) 0 t G()G() I t I t  I t I t      2 I t I t  G O   log  9

10. FCS – autocorrelation analysis I(t)I(t) 0 t G()G() I t I t  I t I t      2 I t I t  G O   log  10

11. FCS – autocorrelation analysis I(t)I(t) 0 t G()G() I t I t  I t I t      2 I t I t  G O   log  11

12. FCS – autocorrelation analysis I(t)I(t) 0 t G()G() I t I t  I t I t      2 I t I t  G O   log  12

13. FCS – autocorrelation analysis G()G() 1/N  1/c log   corr  1/D Fitting the autocorrelation function to appropriate model functions results in properties of the diffusion process the concentration of several species with different hydrodynamic properties 13

14. The Effects of Particle Size on the Autocorrelation Curve 300 um 2 /s 90 um 2 /s 71 um 2 /s Diffusion Constants Fast Diffusion Slow Diffusion Stokes-Einstein Equation: and Monomer --> Dimer Only a change in D by a factor of 2 1/3, or 1.26 14

15. The Effects of Particle Concentration on the Autocorrelation Curve = 4 = 2 Source- Czech Technical University 15

16. Autocorrelation of EGFP & Adenylate Kinase -EGFP *EGFP is a Green Fluoresceine Protein 16 Source:Enrico gratton Lectures

17. Anti-Digoxin Antibody (IgG) Binding to Digoxin-Fluorescein S. Tetin, K. Swift, &, E, Matayoshi, 2003 17

18. Protein Corona on Nano Particle Spherical nanoparticleProtein Molecules 18

19. Nature Nanotechnology8,701–702 (2013) Nano particle Plasma Adsorbed on NPSurface 19

20. Conclusion 20 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

21. 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 21

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30. Expert Supervision By: Dr. C.K.Nandi Associate professor School Of Basic Sciences I.I.T Mandi Presentation By: Kush Kaushik V16016 M.Sc Chemistry I.I.T Mandi Fluorescence Correlation Spectroscopy (FCS) On Protein Corona 30

31. 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. 31

32. 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) 32

33. Slow and Fast Fluctuation-Effect 33

34. Why Confocal Microscope 34

35. The Effects of Particle Size on the Autocorrelation Curve 300 um 2 /s 90 um 2 /s 71 um 2 /s Diffusion Constants Fast Diffusion Slow Diffusion Stokes-Einstein Equation: and Monomer --> Dimer Only a change in D by a factor of 2 1/3, or 1.26 35

36. Time (s) G(  ) EGFP solution EGFP cell EGFP-AK  in the cytosol EGFP-AK in the cytosol Normalized autocorrelation curve of EGFP in solution (), EGFP in the cell ( ), AK1-EGFP in the cell(), AK1  -EGFP in the cytoplasm of the cell(). Autocorrelation of EGFP & Adenylate Kinase -EGFP 36

37. 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. 37

38. 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 -15 L 38

39. 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) = + ƌ N Particle with high Diffusion coefficient shows fast fluctuations. 39

40. Why Confocal Microscope For a Typical Fluorimetre Cuvette(V=100uL, Solution Diluted to 100nM if = 6 x 10^12 Particles Relative Fluctuations( ƌ N Single /N) =.00004% Which is very small to give valid Results 40

41. Why Confocal Microscope 41

42. Why Confocal Microscope Confocal Volume 42

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44. Digoxin-FlIgG (99% bound) Digoxin-Fl Digoxin-FlIgG (50% Bound) Autocorrelation curves: Anti-Digoxin Antibody (IgG) Binding to Digoxin-Fluorescein Binding titration from the autocorrelation analyses: triplet state Kd=12 nM S. Tetin, K. Swift, &, E, Matayoshi, 2003 44

45. 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)I(t) no correlation G()G() t log  I(t)I(t) correlation G()G()  k as k dis t log  45

46. FCCS – model application k as +   k dis G()G() log  46

47. FCCS – model application k as +   k dis G()G() log  47

48. FCCS – model application k as +   k dis G()G() log  48