Template:Virtual Cell Exercises
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'''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. | '''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. | ||
| - | Basic learning goals: | + | ''Basic learning goals:'' |
*To have students do data analysis | *To have students do data analysis | ||
*To become familiar with constructing a simple model in Virtual Cell. | *To become familiar with constructing a simple model in Virtual Cell. | ||
| - | Advanced goals: | + | ''Advanced goals:'' |
Needed: Image stack, image analysis data | Needed: Image stack, image analysis data | ||
Revision as of 17:53, 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 with the Virtual Cell user interface 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
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.
Basic learning goals:
- To have students do data analysis
- To become familiar with constructing a simple model in Virtual Cell.
Advanced goals: 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 (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 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?
