Template:Virtual Cell Exercises - Revision history

Raquell: /* '''Materials Needed''' */

2007-12-20T21:39:48Z

'''Materials Needed'''

← Older revision		Revision as of 21:39, 20 December 2007
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-	BLG: Download excel spreadsheet of fluorescent intensity data ~~(available post workshop)~~.	+	BLG: Download excel spreadsheet of fluorescent intensity data [[mailto:rholmes@uchc.edu\| 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 [[media:FRAP_Homework.xls\|~~ FRAP Homework ~~data coming]]~~ [[mailto:rholmes@uchc.edu\| ~~email~~ Dr.Holmes]] ~~for the 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 spreadsheet of FRAP Homework obtained from [[mailto:rholmes@uchc.edu\| Dr.Holmes]].

	[[image:FiINaROI.jpg]]		[[image:FiINaROI.jpg]]

Raquell at 21:37, 20 December 2007

2007-12-20T21:37:14Z

← Older revision		Revision as of 21:37, 20 December 2007
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	ALG: Image stack, image analysis data		ALG: Image stack, image analysis data
-	To obtain image stack in lab the following are needed:	+	*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).		-Fluorescent microscope -fluorescent probe -labeled cell -camera -image capture and analysis software (e.g. ImageJ).


-	Image stack for extracting values using Image J:	+	*Image stack for extracting values using Image J:
-	*Download and unzip image stack (Coming Soon).	+	**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.	+	***Images are essentially wide field fluorescent images. They were collected with an open pin hole on confocal microscope.

	*Image analysis:		*Image analysis:

Raquell: /* '''Materials Needed:''' */

2007-12-20T21:35:06Z

'''Materials Needed:'''

← Older revision		Revision as of 21:35, 20 December 2007
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	*compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.		*compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.

-	===='''Materials Needed:'''====	+	===='''Materials Needed'''====

	ALG: Image stack, image analysis data		ALG: Image stack, image analysis data

Raquell: /* '''Materials Needed:''' */

2007-12-20T21:34:50Z

'''Materials Needed:'''

← Older revision		Revision as of 21:34, 20 December 2007
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	[[image:FiINaROI.jpg]]		[[image:FiINaROI.jpg]]

-	'''BLG Tasks:'''	+	===='''BLG Tasks'''====

	1. In excel, plot the data as a time series. Compare the plots, normalize based on t/msqi.		1. In excel, plot the data as a time series. Compare the plots, normalize based on t/msqi.
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	2. Compare results to Virtual Cell FRAP simulation created in Tutorial		2. Compare results to Virtual Cell FRAP simulation created in Tutorial
-	What is needed in the simulation for an accurate comparison to experiment?	+
		+	What is needed in the simulation for an accurate comparison to experiment?

Raquell at 21:34, 20 December 2007

2007-12-20T21:34:06Z

← Older revision		Revision as of 21:34, 20 December 2007
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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]'''		'''This exercise draws on a Virtual Cell Tutorial that can be found at the Virtual Cell website [http://vcell.org/login/login.html]'''

-	The first tutorial is based on FRAP. We use the [http://vcell.org/userdocs/Rel/Tutorial_SimpleFRAP.pdf FRAP Tutorial] to become with the Virtual Cell user interface and commands.	+	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.

	===Relevance of FRAP to courses===		===Relevance of FRAP to courses===

Raquell: /* '''Experimental principle:''' */

2007-12-20T21:32:48Z

'''Experimental principle:'''

← Older revision		Revision as of 21:32, 20 December 2007
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	*Data Analysis: image capture, image analysis		*Data Analysis: image capture, image analysis

-	==='''Experimental principle:'''===	+	==='''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.		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.

Raquell: /* '''Materials Needed:''' */

2007-12-20T21:32:32Z

'''Materials Needed:'''

← Older revision		Revision as of 21:32, 20 December 2007
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	'''BLG Tasks:'''		'''BLG Tasks:'''

-	1. In excel, plot the data as a time series. ~~To compare~~ the plots, normalize based on t/msqi.	+	1. In excel, plot the data as a time series. Compare the plots, normalize based on t/msqi.
	*To normalize data across images [[image:FiINaROI.jpg]] vs. (t/msqi)		*To normalize data across images [[image:FiINaROI.jpg]] vs. (t/msqi)


-	2. Compare results to Virtual Cell FRAP simulation	+	2. Compare results to Virtual Cell FRAP simulation created in Tutorial
	What is needed in the simulation for an accurate comparison to experiment?		What is needed in the simulation for an accurate comparison to experiment?

Raquell: /* Relevance of FRAP to courses */

2007-12-20T18:55:27Z

Relevance of FRAP to courses

← Older revision		Revision as of 18:55, 20 December 2007
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	*Data Analysis: image capture, image analysis		*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.	+	==='''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====		====Learning Goals====

Raquell: /* Relevance of FRAP to courses */

2007-12-20T18:48:04Z

Relevance of FRAP to courses

← Older revision		Revision as of 18:48, 20 December 2007
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	*Data Analysis: image capture, image analysis		*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.	+	==='''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):''		''Basic learning goals (BLG):''
	*To have students do data analysis		*To have students do data analysis
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	*compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.		*compare experimental data to multiple biological, computational models i.e. free diffusion, diffusion with binding, immobile fractions, etc.

-	'''Materials Needed:'''	+	===='''Materials Needed:'''====

	ALG: Image stack, image analysis data		ALG: Image stack, image analysis data

Raquell: /* Relevance of FRAP to courses */

2007-12-20T18:44:43Z

Relevance of FRAP to courses

← Older revision		Revision as of 18:44, 20 December 2007
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	BLG: Download excel spreadsheet of fluorescent intensity data (available post workshop).		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 [[media:FRAP_Homework.xls\| FRAP Homework 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 Spreadsheet [[media:FRAP_Homework.xls\| FRAP Homework data coming]] [[mailto:rholmes@uchc.edu\| email Dr.Holmes]] for the data.

	[[image:FiINaROI.jpg]]		[[image:FiINaROI.jpg]]