Fluorescence Correlation Spectroscopy 물리학과/바이오융합기술 바이오융합기술프로젝트I 20050818 차선철 sunchul@sogang.ac.kr

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

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.

2. Setup

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

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

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

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

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

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

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

5. Example Experiment - Diffusion

6. Another Example - Chemical Reaction

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

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 www.rp-photonics.com Nikon Microscopy www.microscopyu.com Mek&Tosj www.mekentosh.com Brownian Motion java animation

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

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

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

Supplement – FRET : Fluorescence Resonance Energy Transfer

Supplement – Student’s DATA