1. Fluorescence Correlation Spectroscopy
물리학과/바이오융합기술
바이오융합기술프로젝트I
차선철

2. Contents What is FCS? Setup Base Theories What’s FCS used for?
Example Experiment - Diffusion
Another Example
Summary

3. 1. What is FCS ?
FCS is
Fluorescence/Fluctuation Correlation Spectroscopy
Suggested 1972, and Renaissance 1990s
Calculate correlation function from time dependant intensity fluctuation of dye molecule by confocal microscopy in single molecule level.

4. 2. Setup

5. By Uncertainty Principle
3. Based Theories – Gaussian Beam
By Uncertainty Principle
Focal Volume
Sample
Lens
laser : λ1
Dichroic Mirror

6. Molecules are Fluorescent
3. Based Theories – Fluorescence
Molecules are Fluorescent λ2 λ1
Fluorescence light : λ2

7. 3. Based Theories – Confocal Microscopy
O.M Resolution limit ~ 200nm
But we want to see single molecule!
-> Confocal Microscopy

8. 3. Based Theories – Correlation Function
Time lag (τ)

9. 4. What’s FCS used for ? From Correlation Function, seeing
Chemical Dynamics,
Intra-molecular /
Inter-molecular
Dynamics,
Diffusion,
E T C…
This Expt.
Study Region

10. 5. Example Experiment - Diffusion
Why does the diffusion create?
-> Brownian Motion !

11. 5. Example Experiment - Diffusion
Changing concentration
-> Changed Correlation Function
Be used only Concentration ~ few nM

12. 5. Example Experiment - Diffusion

13. 6. Another Example - Chemical Reaction

14. 7. Summary We know theory of FCS
: Uncertainty Principle – Gaussian Beam
: Quantum Mechanics – Fluorescence
: Geometric Optics – Confocal Microscopy
: Statistical Mechanics – Correlation function
We use FCS
Study Brownian Motion – Diffusion

15. Reference
P.Schwile and E.Haustein, Fluorescence Correlation Spectroscopy
S.H.Kim, Fluorescence Correlation Spectroscopy
Y.H.Hwang Spectroscopy : Characterization of Material Properties
C.Gell, et al. Handbook of Single Molecule Fluorescence Correlation Spectroscopy
S.Weiss, Fluorescence Spectroscopy of Single Biomolecules
F.Reif, Fundamentals of Statistical and Thermal physics 4th edition
Saleh&Hahaa, Fundamentals of Photonics 4th edition
PerkinElmer, Avalanche Photodiode A User Guide
Hamamatsu, Photomultiplier tubes Basic and Applicaitions
RP Photonics
Nikon Microscopy
Mek&Tosj
Brownian Motion java animation

16. Supplement – Brownian Motion Formula
Molecule Fluctuation Equation of motion : Langevin Equation
Use it -> Given Stokes-Einstein relation
Diffusion coefficient
Intensity

17. Supplement – Correlation function formula
Auto correlation function
Cross correlation function
We use Autocorrelation Function (bottom)

18. Supplement – Photon Counter
Counting A Few Photon
PMT
Photomultiplier tube
APD
Avalanche Photodiode

19. Supplement – FRET
: Fluorescence Resonance Energy Transfer

20. Supplement – Student’s DATA