Template:Virtual Cell Exercises
From Computational Cell Biology
Open Virtual Cell using directions provided.
Create a login name and password. This will last beyond the length of the workshop.
We will examine the FRAP Tutorial to introduce Virtual Cell architecture and commands.
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
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).
Needed: Image stack, image analysis data
To obtain Image stack: 1. Fluorescent microscope, fluorescent probe, labeled cell, camera, image capture software (ImageJ). 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.
Image analysis: 1. Use image analysis software to extract pixel values for fluoresence intensity 2. Download excel spreadsheet of data
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 print out for equations)
To normalize data across images vs. (t/msqi)
Tasks: 1. In excel, plot the data as a time series. To compare the plots, normalize based on t/msqi.
2. Compare results to Virtual Cell FRAP simulation What is needed in the simulation for an accurate comparison to experiment?
