SLIDE 1
Limitations of Fluorescence Correlation Limitations of Fluorescence Correlation Spectroscopy in Complex Situations Spectroscopy in Complex Situations
Jonathan Lam Jonathan Lam
- Dr. Burden
- Dr. Burden
Limitations of Fluorescence Correlation Limitations of Fluorescence - - PowerPoint PPT Presentation
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
SLIDE 2
SLIDE 3
Introduction to FCS Introduction to FCS
Figure 1. Standard experimental set up (Eigen and Rigler, 1994). Illustration of laser focal point on the right.
( ) ( ) ( ) ( ) ( ) [ ]
DC a a g a ag N G
d d
+ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ − + − + =
2 2 1 2 3 2 2 1 3 2 1
1 1 α α τ α τ α γ τ
Analytical Model: Eq. 1
( )
2 1 2 1 3
1 1
− −
⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + =
di di di
g τ κ τ τ τ τ
SLIDE 4
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 Meseth et al. et al. (1999) (1999)
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 analysis analysis
Figure 2. 0.448 fL Ideal Gaussian Beam Profile (Culbertson et al. 2007)
SLIDE 5
The Simulator The Simulator
Single Molecule Diffusion Simulator (SMDS) Single Molecule Diffusion Simulator (SMDS) Python Scripts Python Scripts Simulation Core Simulation Core Parallelized Processing Parallelized Processing Random Walk Random Walk Generates synthetic autocorrelation curve Generates synthetic autocorrelation curve Fitting using analytical model Fitting using analytical model
1E-6 1E-5 1E-4 1E-3 0.01 1.2 1.6 2.0 2.4
- 0.005
0.000 0.005
G (t) Time (s)
RES
SLIDE 6
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 Non-
- Ideal Beam Profile (Two
Ideal Beam Profile (Two Crossed Beams) Crossed Beams)
Analytical Model: Analytical Model:
– – Free and Fixed Parameters ( Free and Fixed Parameters (D Df
f
) )
Results comparison: Results comparison:
– – Diffusion coefficient of the slow Diffusion coefficient of the slow component (D component (Ds
s
) ) – – Mole fraction of the fast Mole fraction of the fast component ( component (a af
f
) ) – – Total concentration ( Total concentration (C Ctotal
total
) )
Figure 4. 0.469 fL crossed beam profile.
SLIDE 7
Fixed Fixed tdx tdx Parameter Parameter
0.0 0.2 0.4 0.6 0.8 1.0 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Concentration (Molecules/μm
3)
Mole Fraction of Fast Component (Simulation Input) Imaxy 913 Imaxy 609 Imaxy 304
- ------ Theoretical
0.0 0.2 0.4 0.6 0.8 1.0 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Concentration (molecules/μm
3)
Mole Fraction of Fast Component (Simulation Input)
Imaxy 913 Imaxy 609 Imaxy 304 Theoretical
Free Free tdx tdx Parameter Parameter
Trends in Total Concentration Due to Different Fitting Routines
Results
SLIDE 8
Dim Slow Component Dim Slow Component
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mole Fraction of Fast Component (Simulation Output) Mole Fraction of Fast Component (Simulation Input)
Imaxy 913 Imaxy 609 Imaxy 304 Theoretical
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mole Fraction of Fast Component (Simulation Output) Mole Fraction of Fast Component (Simulation Input)
Imaxx 913 Imaxx 609 Imaxx 304 Theoretical
Dim Fast Component Dim Fast Component
Trends in Mole Fraction of Fast Component Due to Different Simulation Conditions
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Trends in Diffusion Coefficient Due to Changes in Volume Shape Trends in Diffusion Coefficient Due to Changes in Volume Shape
0.0 0.2 0.4 0.6 0.8 1.0 1E-7 1E-6 1E-5
Diffusion Coefficient (cm
2/s)
Mole Fraction of Fast Component (Simulation Input) Imaxy 913 Imaxy 609 Imaxy 304 Theoretical
0.0 0.2 0.4 0.6 0.8 1.0 1E-9 1E-8 1E-7
Diffusion Coefficient (cm
2/s)
Mole Fraction of Fast Component (Simulation Input) Imaxy 913 Imaxy 609 Imaxy 304 Theoretical
Ideal Gaussian Beam Profile Crossed Beam Profile
SLIDE 10
- All
All D D
f f
values had an error of less than ~1% values had an error of less than ~1% Table 1. Table 1. Effects of Different Intensity Ratios ( Effects of Different Intensity Ratios (α α
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 Slow Dim Fast Dim Fast Dim Slow Dim Slow Parameter Parameter Value of Value of α α
2 2
Fixed Fixed D D
f f
Free Free D D
f f
Fixed Fixed D D
f f
Free Free D D
f f
D D
s s
0.25 0.25 1 1-
- 590%
590% 1 1-
- 562%
562% 0-
- 13%
13% 2 2-
- 100%
100% (cm (cm2
2/s)
/s) 0.5 0.5 0-
- 248%
248% 0-
- 223%
223% 0-
- 32%
32% 1 1-
- 100%
100% 0.75 0.75 0-
- 166%
166% 0-
- 170%
170% 0-
- 76%
76% 0-
- 100%
100% a a
f f
0.25 0.25 0-
- 43%
43% 1 1-
- 70%
70% 0-
- 170%
170% 1 1-
- 75%
75% 0.5 0.5 0-
- 6%
6% 0-
- 20%
20% 0-
- 20%
20% 1 1-
- 60%
60% 0.75 0.75 0-
- 10%
10% 1 1-
- 20%
20% 0-
- 4%
4% 0-
- 30
30 C C 0.25 0.25 1 1-
- 31%
31% 2 2-
- 12%
12% 0-
- 4%
4% 30 30-
- 63%
63% ( (Molec Molec/ /μ μm m3
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%
Results Summary
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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 The non-
- ideal beam profile, if unadjusted for,
ideal beam profile, if unadjusted for, 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.
SLIDE 12
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
SLIDE 13
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