1 15 October 2005 Vermelding onderdeel organisatie Theoretical Testing of Single-Molecule FCS and Two-Color FCCS without Immobilization or Hydrodynamic Focusing Application: 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 P D D r.med.habil. D r.rer.nat . Zeno F öld es -P app 10th International Workshop on FCS and Related Methods, Sapporo, Nov. 26-28, 2007, JAPAN
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
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
2
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
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?
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
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
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)
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)
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)
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
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 .