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15 October 2005
Vermelding onderdeel organisatie
Theoretical Testing of Single-MoleculeFCS and Two-Color FCCS without
Immobilization or Hydrodynamic FocusingApplication: solution, live cell
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
November 27, 2007
2/23
Overview Presentation
1. Problem Description
• Motivation
• Objectives
• Method
2. Modeling
3. Results and Discussions
Introduction – Problem – Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
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3/23
Background - Motivation (1/2)Reentries of a single molecule are a major problem:
• FCS theory could not predict motion of a single molecule
• Experiments showed highly sensitive detection:
Goodwin and Keller (2003) approached fM-range with two-color FCCS
• Multi-parameter fluorescence detection offers an experimental basis for
the collection of both TCSPC data and FCS/FCCS data
� Likelihood estimators
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
4/23
Background - Motivation (2/2)
• Fluctuations are stochastic
• The detected fluorescence signals become digital
• Fluorescence bursts are only detected when single molecules pass through the confocal observation volume
�
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
3
5/23
Problem Statement
• Reentries of a single molecule give raise to fluctuation phenomena
• The molecule diffusing through the focal periphery causes some fluorescence, which is only weakly correlated with that from the sharp focal plane (‘spot’)
Main Question
What happens if the molecule starts near a boundary, i.e. it sits at the border of the confocal observation volume, crosses in and out, and therefore has many reentries but none of them are meaningful?
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
6/23
Objectives
1. Modeling using general tools to solve Markov chains2. Validation of the model using special solutions3. Investigate influence of kinematics to point single-molecule motion
Procedure
1. To develop an accurate hidden Markov model for this challenging application
2. Unravel the position of a single molecule with time
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
4
7/23
Configuration Set-Up
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
8/23
Numerical Modeling
object
Solution generation:
• Reentries depend on motional rates
•
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
5
9/23
Time Step Restrictions
Hence: Ansatz:
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
10/23
Validation Using Special Solutions
Summarizing:
•
•
� Extend this method to meaningful reentries !!!
If the molecule number < 1 then the corresponding time rate of motional single-molecule reentries is given by the diffusion time of the molecule.
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
6
11/23
Verification Using Special Solutions
Numerical model:
•
•
Close-up at the Leading Edge:
Non-meaningful reentries:
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
Meaningful reentries:
fine
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Remember - Solution generation:Reentries depend on motional rates
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
12/23
Verification Using Special Solutions
Numerical model:
•
• HENCE, the meaningful time in the confocal observation volume is
Close-up at the Leading Edge:
Meaningful reentries:
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
Meaningful reentries
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Remember - Solution generation:Reentries depend on motional rates
If the observed N value becomes N < 1 then N stands for the Poisson probabilityof finding a single molecule in the confocalprobe region (arrival of a single molecule). Under this condition, N < 1, <C> = C is theaverage frequency that the confocal probe region contains a single molecule:
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
7
13/23
Verification Using Special Solutions
Numerical model:
•
Close-up at the Leading Edge:
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
Meaningful reentries:
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Remember - Solution generation:Reentries depend on motional rates
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
14/23
Verification Using Special Solutions
Numerical model:
•
Close-up at the Leading Edge:
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
Meaningful reentries:
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Remember - Solution generation:Reentries depend on motional rates
fine
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
8
15/23
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Verification Using an Another Approach
Chance that the Reentering Molecule is not the Original Molecule
As Function of the Time from Last Entry
I take the two-dimensional Poisson probability distribution of finding fluorescent molecules in the detection volume of the bulk phase
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
16/23
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
Chance that the Reentering Molecule is not the Original Molecule
As Function of the Time from Last Entry
Verification Using an Another Approach10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
9
17/23
Definition of Motion Parameters
3D parameters:for the axially-symmetric, cylindricalvolume element in terms of cylindrical polars(q, φφφφ, z) withradial diffusion in space (three-dimensional)
•Density Function of Diffusive Spreading:
n (q, φφφφ, z, t) =
n (q, t)
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
18/23
Kinematic Models
3D parameters:for the axially-symmetric, cylindricalvolume element in terms of cylindrical polars(q, φφφφ, z) withradial diffusion in space (three-dimensional)
•Density Function of Diffusive Spreading:
n (q, φφφφ, z, t) =
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
n (q, t) = f (q, t)
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
10
19/23
Kinematic Models
3D parameters:for the axially-symmetric, cylindricalvolume element in terms of cylindrical polars(q, φφφφ, z) withradial diffusion in space (three-dimensional)
•Density Function of Diffusive Spreading:
n (q, φφφφ, z, t) =
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
n (q, t) = f (q, t)
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
20/23
Summary of Main Results
� Remember - Solution generation: Reentries depend on motional rates
and and
Take a closer look at the experiments done so far
Exact
Analytic solution
first found
Meaningful timeMeaningful reentries
Non-meaningful reentries
Model
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
11
21/23
Summary of Main Results
� Remember - Solution generation:
�Földes-Papp (2007). Fluorescence fluctuation spectroscopic approaches to the study of a
single molecule diffusing in solution and a live cell without systemic drift or convection: a theoretical study. Curr. Pharm. Biotechnol. 8 (5), 261-273.
�Földes-Papp (2007). ‘True’ single-molecule molecule observations by fluorescence
correlation spectroscopy and two-color fluorescence cross-correlation spectroscopy. Exp. Mol. Pathol. 82 (2), 147-155.
�Földes-Papp (2006). What it means to measure a single molecule in a solution by
fluorescence fluctuation spectroscopy. Exp. Mol. Pathol. 80 (3), 209-218.
�Földes-Papp, Baumann, Kinjo, Tamura (2005). Single-phase single-molecule
fluorescence correlation spectroscopy (SPSM-FCS). Distinguished article entry. In: J Fuchs, M Podda (Eds), Encyclopedia of Medical Genomics & Proteomics, Marcel Dekker, New York.
�Földes-Papp (2002). A new dimension for the development of fluorescence-based assays
in solution: from physical principles of FCS detection to biological applications (assays of single molecules in solution). Exp. Biol. Med. 227 (5), 291-300 .
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
22/23
Conclusions
� Földes-Papp (2008). Viral Chip Technology for Genomic Medicine. In: H.F.
Willard, G.S. Ginsburg (Eds.), Handbook of Genomic Medicine, Part I – The Basics, Technolgies. Academic Press, New York. Upcoming in October 2008.
Introduction – Problem – Numerical Modeling – Validation – Kinematic Modeling – Results – Conclusions
PD Dr.med.habil. Dr.rer.nat. Zeno Földes-Papp
10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN