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Fluorescence Correlation Spectroscopy: Parameters, Methods, and Applications

Learn about the principles and techniques of fluorescence correlation spectroscopy, including fluorescence parameters, rotational diffusion, fluorescence quenching, and more. Explore its applications in measuring molecular interactions and dynamics.

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Fluorescence Correlation Spectroscopy: Parameters, Methods, and Applications

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  1. Лекция 6

  2. Флуоресцентная корреляционная спектроскопия

  3. Флуоресцентные параметры и методы • Спектры возбуждения и люминесценции • • Полярность локального окружения, концентрация • люминесциирующих молекул • 2. Анизотропия и поляризация • • вращательная диффузия • 3. Тушение флуоресценции • • «активность» растворителя • • характер локального окружения (локального поля) • 4. Время жизни флуоресценции • • Динамическиеипроцессы (наносекунды) • 5. Резонансный перенос энергии электронного возбуждения • • измерение расстояния между взаимодействующими молекулами • 6. Флуоресцентная микроскопия • • локализация • 7. Флуоресцентная корреляционная спектроскопия • • Трансляционная и вращательная диффузия • • концентрация • • динамика

  4. Диффузия Флуктуации фазы Конформационная динамика Вращательное движение В ФКС Флуктуации дают сигнал Процессы вызывающие флуктуации

  5. Generating Fluctuations By Motion Что мы наблюдаем? 1.Скорость вращения 2. Концентрацию частиц 3.Изменения во флуоресценции частиц во время регистрации, например из-за конформационных переходов Observation Volume Пространство образца

  6. Approximately 1 um3 Defining Our Observation Volume: One- & Two-Photon Excitation. 2 - Photon 1 - Photon Defined by the pinhole size, wavelength, magnification and numerical aperture of the objective Defined by the wavelength and numerical aperture of the objective

  7. 1-photon 2-photon Brad Amos MRC, Cambridge, UK

  8. Data Treatment & Analysis Time Histogram Autocorrelation Autocorrelation Parameters: G(0) & kaction Photon Counting Histogram (PCH) PCH Parameters: <N> & e

  9. Autocorrelation Function Factors influencing the fluorescence signal: C(r,t) is a function of the fluorophore concentration over time. This is the term that contains the “physics” of the diffusion processes kQ = quantum yield and detector sensitivity (how bright is our probe). This term could contain the fluctuation of the fluorescence intensity due to internal processes W(r) describes our observation volume

  10. Calculating the Autocorrelation Function Fluorescence Fluctuation F(t) in photon counts time  Average Fluorescence t t + t

  11. The Autocorrelation Function t3 t5 t4 t2 t1 G(0)  1/N As time (tau) approaches 0 Diffusion

  12. <N> = 2 <N> = 4 The Effects of Particle Concentration on the Autocorrelation Curve

  13. Why Is G(0) Proportional to 1/Particle Number? A Poisson distribution describes the statistics of particle occupancy fluctuations. In a Poissonian system the variance is proportional to the average number of fluctuating species:

  14. What about the excitation (or observation) volume shape?

  15. Effect of Shape on the (Two-Photon) Autocorrelation Functions: For a 2-dimensional Gaussian excitation volume: 1-photon equation contains a 4, instead of 8 For a 3-dimensional Gaussian excitation volume:

  16. Additional Equations: 3D Gaussian Confocor analysis: ... where N is the average particle number, tD is the diffusion time (related to D, tD=w2/8D, for two photon and tD=w2/4D for 1-photon excitation), and S is a shape parameter, equivalent to w/z in the previous equations. Note: The offset of one is caused by a different definition of G() : Triplet state term: ..where T is the triplet state amplitude and tT is the triplet lifetime.

  17. Orders of magnitude (for 1 μM solution, small molecule, water) Volume Device Size(μm) Molecules Time milliliter cuvette 10000 6x1014 104 microliter plate well 1000 6x1011 102 nanoliter microfabrication 100 6x108 1 picoliter typical cell 10 6x105 10-2 femtoliter confocal volume 1 6x102 10-4 attoliter nanofabrication 0.1 6x10-1 10-6

  18. The Effects of Particle Size on the Autocorrelation Curve Diffusion Constants 300 um2/s 90 um2/s 71 um2/s Slow Diffusion Fast Diffusion Stokes-Einstein Equation: and Monomer --> Dimer Only a change in D by a factor of 21/3, or 1.26

  19. Dsolution = 3.3 Dnucleus FCS inside living cells Two-Photon Spot Correlation Analysis Coverslip objective Measure the diffusion coefficient of Green Fluorescent Protein (GFP) in aqueous solution in inside the nucleus of a cell.

  20. Autocorrelation Adenylate Kinase -EGFP Chimeric Protein in HeLa Cells Examples of different Hela cells transfected with AK1-EGFP Fluorescence Intensity Examples of different Hela cells transfected with AK1b -EGFP Qiao Qiao Ruan, Y. Chen, M. Glaser & W. Mantulin Dept. Biochem & Dept Physics- LFD Univ Il, USA

  21. Autocorrelation of EGFP & Adenylate Kinase -EGFP EGFP-AK in the cytosol G(t) EGFP-AKb in the cytosol EGFPsolution EGFPcell Time (s) Normalized autocorrelation curve of EGFP in solution (•), EGFP in the cell (•), AK1-EGFP in the cell(•), AK1b-EGFP in the cytoplasm of the cell(•).

  22. Autocorrelation of Adenylate Kinase –EGFP on the Membrane Clearly more than one diffusion time A mixture of AK1b-EGFP in the cytoplasm and membrane of the cell.

  23. Multiple Species G(0)sample is no longer g/N ! Case 1: Species vary by a difference in diffusion constant, D. Autocorrelation function can be used: (2D-Gaussian Shape) ! fi is the fractional fluorescence intensity of species i.

  24. Antibody - Hapten Interactions Binding site Binding site carb2 Digoxin: a cardiac glycoside used to treat congestive heart failure. Digoxin competes with potassium for a binding site on an enzyme, referred to as potassium-ATPase. Digoxin inhibits the Na-K ATPase pump in the myocardial cell membrane. Mouse IgG: The two heavy chains are shown in yellow and light blue. The two light chains are shown in green and dark blue..J.Harris, S.B.Larson, K.W.Hasel, A.McPherson, "Refined structure of an intact IgG2a monoclonal antibody", Biochemistry 36: 1581, (1997).

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

  26. Two Binding Site Model IgG•2Ligand-Fl + Ligand-Fl IgG + 2 Ligand-Fl IgG•Ligand-Fl 50% quenching Kd IgG•Ligand No quenching IgG•2Ligand [Ligand]=1, G(0)=1/N, Kd=1.0

  27. Multiple Species G(0)sample is no longer g/N ! Case 1: Species vary by a difference in diffusion constant, D. Autocorrelation function can be used: (2D-Gaussian Shape) ! fi is the fractional fluorescence intensity of species i.

  28. Antibody - Hapten Interactions Binding site Binding site carb2 Digoxin: a cardiac glycoside used to treat congestive heart failure. Digoxin competes with potassium for a binding site on an enzyme, referred to as potassium-ATPase. Digoxin inhibits the Na-K ATPase pump in the myocardial cell membrane. Mouse IgG: The two heavy chains are shown in yellow and light blue. The two light chains are shown in green and dark blue..J.Harris, S.B.Larson, K.W.Hasel, A.McPherson, "Refined structure of an intact IgG2a monoclonal antibody", Biochemistry 36: 1581, (1997).

  29. Case 2: Species vary by a difference in brightness assuming that The quantity G(0) becomes the only parameter to distinguish species, but we know that: The autocorrelation function is not suitable for analysis of this kind of data without additional information. We need a different type of analysis

  30. Photon Counting Histogram (PCH) To resolve species from differences in their molecular brightness Aim: Molecular brightness ε: The average photon count rate of a single fluorophore where p(k) is the probability of observing k photon counts PCH: probability distribution function p(k) Single Species: Note: PCH is Non-Poissonian! Sources of Non-Poissonian Noise • Detector Noise • Diffusing Particles in an Inhomogeneous Excitation Beam* • Particle Number Fluctuations* • Multiple Species*

  31. PCH Example: Differences in Brightness frequency (en=1.0) (en=2.2) (en=3.7) Increasing Brightness Photon Counts

  32. Single Species PCH: Concentration 5.5 nM Fluorescein Fit: e = 16,000 cpsm N = 0.3 550 nM Fluorescein Fit: e = 16,000 cpsm N = 33 As particle concentration increases the PCH approaches a Poisson distribution

  33. Photon Counting Histogram: Multispecies Binary Mixture: Molecular Brightness Concentration Snapshots of the excitation volume Intensity Time

  34. Sample 1: fewer but brighter fluors (10 nM Rhodamine) Sample 3: The mixture Photon Counting Histogram: Multispecies Sample 2: many but dim (23 nM fluorescein at pH 6.3) The occupancy fluctuations for each specie in the mixture becomes a convolution of the individual specie histograms. The resulting histogram is then broader than expected for a single species.

  35. Resolve a protein mixture with a brightness ratio of two Alcohol dehydrogenase labeling experiments Mixture of singly or doubly labeled proteins Singly labeled proteins + ! Both species have same • color • fluorescence lifetime • diffusion coefficient • polarization kcpsm kcpsm

  36. ECFP: EYFP: A + A A A Apparent Brightness A ε 2ε ε 2ε ε A ε ε 0 ε 2ε A B B B B B Distinguish Homo- and Hetero-interactions in living cells • single detection channel experiment • distinguish between CFP and YFP by excitation (not by emission)! • brightness of CFP and YFP is identical at 905nm (with the appropriate filters) • you can choose conditions so that the brightness is not changed by FRET between CFP and YFP • determine the expressed protein concentrations of each cell!

  37. Two Channel Detection: Cross-correlation Sample Excitation Volume Beam Splitter • Increases signal to noise by isolating correlated signals. • Corrects for PMT noise Detector 1 Detector 2 Each detector observes the same particles

  38. Removal of Detector Noise by Cross-correlation Detector 1 11.5 nM Fluorescein Detector 2 Detector after-pulsing Cross-correlation

  39. Calculating the Cross-correlation Function Detector 1: Fi time  t + t t Detector 2: Fj time

  40. Cross-correlation Calculations One uses the same fitting functions you would use for the standard autocorrelation curves. Thus, for a 3-dimensional Gaussian excitation volume one uses: G12 is commonly usedto denote the cross-correlation and G1 and G2 for the autocorrelation of the individual detectors. Sometimes you will see Gx(0) or C(0) used for the cross-correlation.

  41. Two-Color Cross-correlation The cross-correlation ONLY if particles are observed in both channels Sample Green filter Red filter Each detector observes particles with a particular color The cross-correlation signal: Only the green-red molecules are observed!!

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