Limitations of Fluorescence Correlation Limitations of Fluorescence - PowerPoint PPT Presentation

Limitations of Fluorescence Correlation Limitations of Fluorescence Correlation Spectroscopy in Complex Situations Spectroscopy in Complex Situations Jonathan Lam Jonathan Lam Dr. Burden Dr. Burden Summer 2008 Summer 2008

Outline Outline Introduction to FCS Introduction to FCS A. A. Context of the Project Context of the Project B. B. The Simulator The Simulator C. C. Conditions to Consider Conditions to Consider D. D. Results Results E. E. Conclusions Conclusions F. F. Future Projects Future Projects G. G.

Introduction to FCS Introduction to FCS Figure 1. Standard experimental set up (Eigen and Rigler, 1994). Illustration of laser focal point on the right. ( ) ( ) ( ) ⎛ ⎞ γ α 2 τ + α 2 − τ 1 ( ) ag a g ⎜ ⎟ Analytical Model: Eq. 1 τ = + 1 3 1 2 3 2 d d G DC ⎜ [ ] ⎟ ( ) α + α − 2 1 N ⎝ ⎠ a a 1 2 1 − − 1 ⎛ + ⎞ ⎛ + ⎞ τ τ 2 ( ) ⎜ ⎟ ⎜ ⎟ τ = 1 1 g ⎜ ⎟ ⎜ ⎟ 3 τ κ 2 τ di ⎝ ⎠ ⎝ ⎠ di di

Context of the Project Context of the Project Limitations of FCS: Limitations of FCS: – Type of system being – Type of system being analyzed analyzed – Shape of the observation – Shape of the observation volume volume – – The analytical model The analytical model Literature: Literature: – Meseth (1999) – Meseth et al. (1999) et al. Measurement accuracy of Measurement accuracy of diffusion, concentration, diffusion, concentration, and mole fraction and mole fraction Goal: Goal: – Characterize trends in the – Characterize trends in the resolution limits of FCS resolution limits of FCS – Explore novel methods of – Explore novel methods of Figure 2. 0.448 fL Ideal Gaussian analysis analysis Beam Profile (Culbertson et al. 2007)

The Simulator The Simulator Single Molecule Diffusion Simulator (SMDS) Single Molecule Diffusion Simulator (SMDS) Python Scripts � � Simulation Core Simulation Core Python Scripts Parallelized Processing Parallelized Processing Random Walk Random Walk Generates synthetic autocorrelation curve Generates synthetic autocorrelation curve Fitting using analytical model Fitting using analytical model 2.4 2.0 G (t) 1.6 1.2 1E-6 1E-5 1E-4 1E-3 0.01 Time (s) 0.005 0.000 RES -0.005

Conditions to Consider Conditions to Consider Simulation Conditions: Simulation Conditions: – – Two- Two -Component Diffusion Component Diffusion (Diffusion coefficients fixed) (Diffusion coefficients fixed) Different Intensity Ratios (dim Different Intensity Ratios (dim fast or slow component) fast or slow component) Different Mole Fractions (ranging Different Mole Fractions (ranging from 0.01 – from 0.01 – 0.99) 0.99) – Non- -Ideal Beam Profile (Two Ideal Beam Profile (Two – Non Crossed Beams) Crossed Beams) Analytical Model: Analytical Model: – Free and Fixed Parameters (D D f ) – Free and Fixed Parameters ( ) f Results comparison: Results comparison: – Diffusion coefficient of the slow – Diffusion coefficient of the slow component (D s component (D ) ) s Figure 4. 0.469 fL crossed beam profile. – – Mole fraction of the fast Mole fraction of the fast component (a a f ) component ( ) f – Total concentration (C C total ) – Total concentration ( ) total

Results Trends in Total Concentration Due to Different Fitting Routines 1.00 1.00 Imaxy 913 Imaxy 913 Imaxy 609 Imaxy 609 Imaxy 304 Imaxy 304 0.95 0.95 Theoretical ------- Theoretical 3 ) 3 ) Concentration (Molecules/ μ m Concentration (molecules/ μ m 0.90 0.90 0.85 0.85 0.80 0.80 0.75 0.75 0.70 0.70 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Mole Fraction of Fast Component (Simulation Input) Mole Fraction of Fast Component (Simulation Input) Free Free tdx tdx Parameter Parameter Fixed tdx tdx Parameter Fixed Parameter

Trends in Mole Fraction of Fast Component Due to Different Simulation Conditions Mole Fraction of Fast Component (Simulation Output) 1.0 1.0 Mole Fraction of Fast Component (Simulation Output) Imaxx 913 Imaxy 913 Imaxx 609 0.9 0.9 Imaxy 609 Imaxx 304 Imaxy 304 0.8 0.8 Theoretical Theoretical 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Mole Fraction of Fast Component (Simulation Input) Mole Fraction of Fast Component (Simulation Input) Dim Slow Component Dim Slow Component Dim Fast Component Dim Fast Component

Trends in Diffusion Coefficient Due to Changes in Volume Shape Trends in Diffusion Coefficient Due to Changes in Volume Shape 1E-5 Imaxy 913 Imaxy 609 1E-7 Imaxy 304 Theoretical 2 /s) 2 /s) Diffusion Coefficient (cm Diffusion Coefficient (cm 1E-6 1E-8 Imaxy 913 Imaxy 609 Imaxy 304 1E-9 Theoretical 1E-7 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 Mole Fraction of Fast Component (Simulation Input) Mole Fraction of Fast Component (Simulation Input) Ideal Gaussian Beam Profile Crossed Beam Profile

Results Summary Effects of Different Intensity Ratios ( α α ) Table 1. Effects of Different Intensity Ratios ( ) Table 1. 2 2 Range of Error over a Range of Error over a Ideal Gaussian Ideal Gaussian Non Non- -Ideal (Crossed Beams) Ideal (Crossed Beams) Dim Slow Dim Fast Dim Slow Dim Slow Dim Fast Dim Slow Parameter Value of α Fixed D D Free D D Fixed D D Free D D Parameter Value of α Fixed Free Fixed Free 2 2 f f f f f f f f D D 0.25 0.25 1- 1 -590% 590% 1- 1 -562% 562% 0 0- -13% 13% 2- 2 -100% 100% s s (cm 2 (cm 2 /s) /s) 0.5 0.5 0 0- -248% 248% 0- 0 -223% 223% 0- 0 -32% 32% 1- 1 -100% 100% 0.75 0- -166% 166% 0- -170% 170% 0- -76% 76% 0- -100% 100% 0.75 0 0 0 0 a 0.25 0- -43% 43% 1- -70% 70% 0- -170% 170% 1- -75% 75% a 0.25 0 1 0 1 f f 0.5 0- -6% 6% 0- -20% 20% 0- -20% 20% 1- -60% 60% 0.5 0 0 0 1 0.75 0.75 0- 0 -10% 10% 1 1- -20% 20% 0- 0 -4% 4% 0- 0 -30 30 C 0.25 1- -31% 31% 2- -12% 12% 0- -4% 4% 30- -63% 63% C 0.25 1 2 0 30 (Molec ( Molec/ / μ μ m m 3 3 ) ) 0.5 0.5 1- 1 -3% 3% 1- 1 -4% 4% 1- 1 -2% 2% 27- 27 -35% 35% 0.75 0.75 1- 1 -2% 2% 1 1- -2% 2% 1 1- -2% 2% 21- 21 -35% 35% •All All D D values had an error of less than ~1% • values had an error of less than ~1% f f

Conclusion Conclusion Using an ideal Gaussian beam profile, the Using an ideal Gaussian beam profile, the analytical model is more robust than expected. analytical model is more robust than expected. – The model properly functions when diffusion is a free – The model properly functions when diffusion is a free parameter parameter – Mole fractions can be resolved with reasonable – Mole fractions can be resolved with reasonable accuracy in a range of conditions accuracy in a range of conditions – Concentration measurements have greater limitations – Concentration measurements have greater limitations that reported that reported The non- -ideal beam profile, if unadjusted for, ideal beam profile, if unadjusted for, The non severally limits the application of FCS. severally limits the application of FCS. There are clear limitations in FCS that require There are clear limitations in FCS that require new methods of analysis to overcome. new methods of analysis to overcome.

Future Projects Future Projects Completing trials on combinations of Completing trials on combinations of conditions conditions – Variable diffusion coefficients – Variable diffusion coefficients Publication writing Publication writing Applying NFCS analysis Applying NFCS analysis

Acknowledgements Acknowledgements Wheaton College and the Chemistry Department Wheaton College and the Chemistry Department The Wheaton College Alumni Association The Wheaton College Alumni Association Michael Culbertson Michael Culbertson Dr Dorothy Chappell, Dean Dr Dorothy Chappell, Dean Dr. Stanton Jones, Provost Dr. Stanton Jones, Provost National Science Foundation National Science Foundation American Chemical Society American Chemical Society