Template:Virtual Cell Exercises

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Open Virtual Cell using directions provided.
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'''This exercise draws on a Virtual Cell Tutorial that can be found at the Virtual Cell website [http://vcell.org/login/login.html]'''
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Create a login name and password. This will last beyond the length of the workshop.
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The first tutorial is based on FRAP. We use the [http://vcell.org/userdocs/Rel/Tutorial_SimpleFRAP.pdf FRAP Tutorial] to become familiar with the Virtual Cell user interface and commands. What is outlined below is the experimental context and homework assignment that accompanies the tutorial.
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We will examine the [http://www.vcell.org/login/frap.pdf FRAP Tutorial] to introduce Virtual Cell architecture and commands.
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===Relevance of FRAP to courses===
===Relevance of FRAP to courses===
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'''Concepts'''
'''Concepts'''
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Biology: diffusion, compartments
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*Biology: diffusion, compartments
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Experiments: time series, fluorescent microscopy, wave lengths, lasers
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Data Analysis: image capture, image analysis
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*Experiments: time series, fluorescent microscopy, wave lengths, lasers
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*Data Analysis: image capture, image analysis
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==='''Experimental principle'''===
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Fluorescence Recovery After Photobleaching (FRAP) is a fluorescent optical technique used to measure the dynamics of a population of molecules over time and chemical changes of molecular species. The fluorescently labeled molecules are visualized through an epifluorescent or confocal microscope using low light excitation. The excitation light is focused onto a small region of molecules and pulsed to a high intensity in order to photobleach the fluorophore within the illuminated region. A blackened area of photobleached molecules surrounded by fluorescently labeled molecules that are not photobleached will result from the high intensity light. The molecules that are not photobleached will diffuse, providing they can, into this region. The blackened area will gradually increase in intensity (recover) over time as the molecules diffuse.
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====Learning Goals====
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''Basic learning goals (BLG):''
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*To have students do data analysis
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*To become familiar with constructing a simple model in Virtual Cell.
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''Advanced learning goals (ALG):''
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*extract data values from image files
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*compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.
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===='''Materials Needed'''====
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ALG: Image stack, image analysis data
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*To obtain image stack in lab the following are needed:
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-Fluorescent microscope  -fluorescent probe  -labeled cell  -camera  -image capture and analysis software (e.g. ImageJ).
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*Image stack for extracting values using Image J:
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**Download and unzip image stack (Coming Soon).
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***Images are essentially wide field fluorescent images. They were collected with an open pin hole on confocal microscope.
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Photobleaching experimental principle: Fluorescence Recovery After Photobleaching (FRAP) is a fluorescent optical technique used to measure the dynamics of a molecule over time and chemical changes of molecular species. The fluorescently labeled molecules are visualized through an epifluorescent or confocal microscope using low light excitation. The excitation light is focused onto a small region of molecules and pulsed to a high intensity in order to photobleach the fluorophore within the illuminated region. A blackened area of photobleached molecules surrounded by fluorescently labeled molecules that are not photobleached will result from the high intensity light. The molecules that are not photobleached will diffuse, providing they can, into this region. The blackened area will gradually increase in intensity over time as the molecules diffuse in (Virtual Cell FRAP Tutorial).
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*Image analysis:  
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**Use image analysis software to extract pixel values for fluoresence intensity  
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Needed: Image stack, image analysis data
 
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To obtain Image stack:  
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BLG: Download excel spreadsheet of fluorescent intensity data [[mailto:rholmes@uchc.edu| email Dr.Holmes]].
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1. Fluorescent microscope, fluorescent probe, labeled cell, camera, image capture software (ImageJ).
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2. Download and unzip image stack (available after workshop). Images are essentially wide field fluorescent images. They were collected with an open pin hole on confocal microscope.
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Image analysis:  
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Data Set: The first postbleach image is at 0 time ( t=0). For each set of data (i=1,..,4), we give the number of microns squared in the bleach region (msqi), and the average pre-bleach fluorescence within the same region, [Fi(-)]. See spreadsheet of FRAP Homework obtained from [[mailto:rholmes@uchc.edu| Dr.Holmes]].
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1. Use image analysis software to extract pixel values for fluoresence intensity
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2. Download excel spreadsheet of data (available post workshop).
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Data Set: The first postbleach image is at 0 time ( t=0). For each set of data (i=1,..,4), we give the number of microns squared in the bleach region (msqi), and the average pre-bleach fluorescence within the same region.  [Fi(-)]. 
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[[image:FiINaROI.jpg]]
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'''(See print out for equations)'''   
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===='''BLG Tasks'''====
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1. In excel, plot the data as a time series. Compare the plots, normalize based on t/msqi.
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*To normalize data across images  [[image:FiINaROI.jpg]]  vs.    (t/msqi)
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To normalize data across images      vs.    (t/msqi)
 
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'''Tasks:'''
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2. Compare results to Virtual Cell FRAP simulation created in Tutorial
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1. In excel, plot the data as a time series. To compare the plots, normalize based on t/msqi.
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2. Compare results to Virtual Cell FRAP simulation
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What is needed in the simulation for an accurate comparison to experiment?
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What is needed in the simulation for an accurate comparison to experiment?
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Latest revision as of 21:39, 20 December 2007

This exercise draws on a Virtual Cell Tutorial that can be found at the Virtual Cell website [1]

The first tutorial is based on FRAP. We use the FRAP Tutorial to become familiar with the Virtual Cell user interface and commands. What is outlined below is the experimental context and homework assignment that accompanies the tutorial.

Contents

Relevance of FRAP to courses

Course Relevance: Cell Biology, Biochemistry

Concepts

  • Biology: diffusion, compartments
  • Experiments: time series, fluorescent microscopy, wave lengths, lasers
  • Data Analysis: image capture, image analysis

Experimental principle

Fluorescence Recovery After Photobleaching (FRAP) is a fluorescent optical technique used to measure the dynamics of a population of molecules over time and chemical changes of molecular species. The fluorescently labeled molecules are visualized through an epifluorescent or confocal microscope using low light excitation. The excitation light is focused onto a small region of molecules and pulsed to a high intensity in order to photobleach the fluorophore within the illuminated region. A blackened area of photobleached molecules surrounded by fluorescently labeled molecules that are not photobleached will result from the high intensity light. The molecules that are not photobleached will diffuse, providing they can, into this region. The blackened area will gradually increase in intensity (recover) over time as the molecules diffuse.

Learning Goals

Basic learning goals (BLG):

  • To have students do data analysis
  • To become familiar with constructing a simple model in Virtual Cell.

Advanced learning goals (ALG):

  • extract data values from image files
  • compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.

Materials Needed

ALG: Image stack, image analysis data

  • To obtain image stack in lab the following are needed:

-Fluorescent microscope -fluorescent probe -labeled cell -camera -image capture and analysis software (e.g. ImageJ).


  • Image stack for extracting values using Image J:
    • Download and unzip image stack (Coming Soon).
      • Images are essentially wide field fluorescent images. They were collected with an open pin hole on confocal microscope.
  • Image analysis:
    • Use image analysis software to extract pixel values for fluoresence intensity


BLG: Download excel spreadsheet of fluorescent intensity data [email Dr.Holmes].

Data Set: The first postbleach image is at 0 time ( t=0). For each set of data (i=1,..,4), we give the number of microns squared in the bleach region (msqi), and the average pre-bleach fluorescence within the same region, [Fi(-)]. See spreadsheet of FRAP Homework obtained from [Dr.Holmes].

FiINaROI.jpg

BLG Tasks

1. In excel, plot the data as a time series. Compare the plots, normalize based on t/msqi.

  • To normalize data across images FiINaROI.jpg vs. (t/msqi)


2. Compare results to Virtual Cell FRAP simulation created in Tutorial

What is needed in the simulation for an accurate comparison to experiment?

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