foldes papp single-molecules-solution_live_cell

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

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Overview Presentation

1. Problem Description

• Motivation

• Objectives

• Method

2. Modeling

3. Results and Discussions

Introduction – Problem – Modeling – Results – Conclusions



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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



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

�




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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?




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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




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7/23

Configuration Set-Up




8/23

Numerical Modeling

object

Solution generation:

• Reentries depend on motional rates

•




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9/23

Time Step Restrictions

Hence: Ansatz:




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.




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11/23

Verification Using Special Solutions

Numerical model:

•

•

Close-up at the Leading Edge:

Non-meaningful reentries:


Meaningful reentries:

fine


Remember - Solution generation:Reentries depend on motional rates


12/23


Numerical model:

•

• HENCE, the meaningful time in the confocal observation volume is




Meaningful reentries



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:


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Numerical model:

•







14/23


Numerical model:

•






fine


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15/23


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


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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

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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)




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Kinematic Models






n (q, t) = f (q, t)


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Kinematic Models






n (q, t) = f (q, t)


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




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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 .




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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.




foldes papp single-molecules-solution_live_cell

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