Template:Virtual Cell Exercises
From Computational Cell Biology
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 (available post workshop).
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 FRAP Homework data coming [email Dr.Holmes] for the data.
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
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?
