Microscopy Biomedical Imaging

of 109

  • date post

    14-Apr-2018
  • Category

    Documents

  • view

    214
  • download

    0

Embed Size (px)

Transcript of Microscopy Biomedical Imaging

  • 7/30/2019 Microscopy Biomedical Imaging

    1/109

    Detection of Non-Brownian Diffusion in the Cell Membrane inSingle Molecule Tracking

    Behind the hop diffusionBased on: Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking

    Ken Ritchie, Xiao-Yuan Shan, Junko Kondo, Kokoro Iwasawa, Takahiro Fujiwara, and Akihiro Kusumi

    Biophysical Journal Volume 88 March 2005

    Rodrigo Rojas Moraleda

    January 2011

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 1/53

  • 7/30/2019 Microscopy Biomedical Imaging

    2/109

    Outline

    1 Objectives

    2 Introduction

    3 Background

    4 Simulation

    5 Model Evaluation

    6 Discussion

    7 Glosary

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 2/53

  • 7/30/2019 Microscopy Biomedical Imaging

    3/109

    Outline

    1 Objectives

    2 Introduction

    3 Background

    4 Simulation

    5 Model Evaluation

    6 Discussion

    7 Glosary

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 3/53

  • 7/30/2019 Microscopy Biomedical Imaging

    4/109

    Objectives

    This work presents the use of simulation to create a baseline from which to study andanalyze:

    Confined diffusion phenomena in plasma membrane.

    The relationship between the data acquisition rate and the interpretation of thediffusion.

    Using a Monte Carlo algorithm, molecules undergoing simple brownian, totallyconfined, and hop diffusion, where simulated. Next, the characteristics determined areexperimentally examined.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 4/53

  • 7/30/2019 Microscopy Biomedical Imaging

    5/109

    Hypothesis

    The implicit assumption hypothesis of this work is:

    Is possible to establish a relationship between the data acquisition rate

    (observation frame rate) and the interpretation of the diffusioncharacteristics of individual particles/molecules

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 5/53

  • 7/30/2019 Microscopy Biomedical Imaging

    6/109

    Outline

    1 Objectives

    2 Introduction

    3 Background

    4 Simulation

    5 Model Evaluation

    6 Discussion

    7 Glosary

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 6/53

  • 7/30/2019 Microscopy Biomedical Imaging

    7/109

    Diffusion in plasma membrane Why?

    Many cellular processes, such as signaling processes, involve the interaction of severalindividual molecules that must come together to transmit information across the

    plasma membrane to the cell interior. Hence, it is of great importance to understandthe mechanism by which the motion of transmembrane and membrane-associatedmolecules is regulated in the cell membrane.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 7/53

  • 7/30/2019 Microscopy Biomedical Imaging

    8/109

    Diffusion in plasma membrane Difficulty

    However, in the cells, molecular behavior is very inhomogeneous, even molecules ofsingle species interact stochastically with distinct molecules or cellular structures in avariety of local environments. Furthermore, molecular interactions are by naturestochastic.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 8/53

  • 7/30/2019 Microscopy Biomedical Imaging

    9/109

    Outline

    1 Objectives

    2 Introduction

    3 Background

    4 Simulation

    5 Model Evaluation

    6 Discussion

    7 Glosary

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 9/53

  • 7/30/2019 Microscopy Biomedical Imaging

    10/109

    Background

    Main features about this simulation:

    Particles were represented by a Gaussian intensity of 250nm width.

    Particle motion were simulated in time steps of 1s.

    Each captured frame contains the sum of individual 1s particle motions.

    The pixel resolution in the image acquisition was simulated to 40nm/pixel.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 10/53

  • 7/30/2019 Microscopy Biomedical Imaging

    11/109

    Background

    Main features about this simulation:

    Particles were represented by a Gaussian intensity of 250nm width.

    Particle motion were simulated in time steps of 1s.

    Each captured frame contains the sum of individual 1s particle motions.

    The pixel resolution in the image acquisition was simulated to 40nm/pixel.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 10/53

    B k d

  • 7/30/2019 Microscopy Biomedical Imaging

    12/109

    Background

    Main features about this simulation:

    Particles were represented by a Gaussian intensity of 250nm width.

    Particle motion were simulated in time steps of 1s.

    Each captured frame contains the sum of individual 1s particle motions.

    The pixel resolution in the image acquisition was simulated to 40nm/pixel.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 10/53

    B k d

  • 7/30/2019 Microscopy Biomedical Imaging

    13/109

    Background

    Main features about this simulation:

    Particles were represented by a Gaussian intensity of 250nm width.

    Particle motion were simulated in time steps of 1s.

    Each captured frame contains the sum of individual 1s particle motions.

    The pixel resolution in the image acquisition was simulated to 40nm/pixel.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 10/53

    B k d

  • 7/30/2019 Microscopy Biomedical Imaging

    14/109

    Background

    Main features about this simulation:

    Particles were represented by a Gaussian intensity of 250nm width.

    Particle motion were simulated in time steps of 1s.

    Each captured frame contains the sum of individual 1s particle motions.

    The pixel resolution in the image acquisition was simulated to 40nm/pixel.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 10/53

    B k d

  • 7/30/2019 Microscopy Biomedical Imaging

    15/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of a single molecules.

    Calculate the mean square displacement (MSD) for every lag time ( )

    Plotting it as a function of corresponding

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 11/53

    Background

  • 7/30/2019 Microscopy Biomedical Imaging

    16/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of a single molecules.

    Calculate the mean square displacement (MSD) for every lag time ( )

    Plotting it as a function of corresponding

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 11/53

    Background P

  • 7/30/2019 Microscopy Biomedical Imaging

    17/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of a single molecules.

    Calculate the mean square displacement (MSD) for every lag time ( )

    Plotting it as a function of corresponding

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 11/53

    Background P

  • 7/30/2019 Microscopy Biomedical Imaging

    18/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of a single molecules.

    Calculate the mean square displacement (MSD) for every lag time ( )

    Plotting it as a function of corresponding

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 11/53

    Background

  • 7/30/2019 Microscopy Biomedical Imaging

    19/109

    Background

    Figure: a kernel image of the diffusion probe was taken from the first frame of the video.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 12/53

    Background Process

  • 7/30/2019 Microscopy Biomedical Imaging

    20/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of a single molecules.

    Calculate the mean square displacement (MSD) for every lag time ( )

    Plotting it as a function of corresponding

    MSD is determined byThe position information (r1(x1, y1), r2(x2, y2), r3(x3, y3), . . .) of single moleculesin the recorded trajectory at a fixed acquisition time, t .

    Lag time is given as n = nt where n is the number of lags between two steps.

    Rodrigo Rojas Moraleda Detection of Non-Brownian Diffusion in the Cell Membrane in Single Molecule Tracking 13/53

    Background Process

  • 7/30/2019 Microscopy Biomedical Imaging

    21/109

    Background Process

    To characterize the particle motion mode:

    Obtain the trajectory of