FrapCalc
Analyze FRAP curves and get parameter by curve fitting (documentation.). Lecture notes are also available ( Lecture Notes (Apr. 2010))
Author
Kota Miura
Centre for Molecular and Cellular Imaging (CMCI)
EMBL Heidelberg
miura at embl dot de
Tel: +49 6221 387 404
Questions & Comments
? Please post in the commenting section below at the bottom of this page
Source
Released under the GNU General Public License.
Requires
IgorPro ver 5 or higher (IgorPro, Wavemetrics).
Installation
Download K_FRAPcalcV9k.ipf to a place where you can access easily. Open the file in IgorPro and compile.
Description
Analyze FRAP curves to derive recovery parameters. More details are in the documentation.
Features
- Import FRAP measurement tab-delimited text files of Zeiss data format (6 different data structures)
Leica data format (7 different data structures) - Three different ways of normalizing FRAP curve - Curve fitting by single and double exponential formula and 2 diffusion formula. - Correction for the acquisition bleaching.
- Output of half-max, diffusion coefficient, mobile/immobile fractions.
- Weighting for the fitting.
- Evaluation of goodness of fit by chi-square and gamma function.
- Graphing of the fitted curve and estimation curve without acquisition bleaching.
- Batch Fitting
Papers + Misc
Papers using this script
- Runz, H. et al., 2006. Sterols regulate ER-export dynamics of secretory cargo protein ts-O45-G. The EMBO journal, 25(13), p.2953-65. Accessed February 7, 2012.
- Atlason, P.T. et al., 2007. N-Methyl-D-aspartate (NMDA) receptor subunit NR1 forms the substrate for oligomeric assembly of the NMDA receptor. The Journal of biological chemistry, 282(35), p.25299-307. Accessed February 7, 2012.
- Pooler, A.M. & McIlhinney, R.A.J., 2007. Lateral diffusion of the GABAB receptor is regulated by the GABAB2 C terminus. The Journal of biological chemistry, 282(35), p.25349-56. Accessed February 7, 2012.
- Hoppa, M.B. et al., 2009. Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules. Cell metabolism, 10(6), p.455-65. Accessed February 7, 2012.
- Treeck, M. et al., 2009. Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process. PLoS pathogens, 5(3), p.e1000322. Accessed February 7, 2012.
- Bosch-Presegué, L. et al., 2011. Stabilization of Suv39H1 by SirT1 is part of oxidative stress response and ensures genome protection. Molecular cell, 42(2), p.210-23. Accessed January 30, 2012.
- Caillier, B. et al., 2011. QRS widening and QT prolongation under bupropion: a unique cardiac electrophysiological profile. Fundamental & clinical pharmacology. Accessed February 7, 2012.
- Díaz, A. et al., 2011. Processivity and subcellular localization of glycogen synthase depend on a non-catalytic high affinity glycogen-binding site. The Journal of biological chemistry, 286(21), p.18505-14. Accessed February 7, 2012.
… searches were done by google hits, so if YOURS is missing please tell me!
Web Back Links
-
- MatLab-migrated version of FRAPCalc.
- Dissertation using FRAPCalc
History
- 05-9-9
- 1st version
- 07-3-28
- 9th version
- 07-8-27 ver 9a
- Added Leica new file format importing (csv format)
- Made K_SoumpasisHalfLife2(fitwave,amplitude) for calculateing Half max. Previous one had problem when point 1 is larger than 0.5 ? Changes for the texts in the graph (Half max value added to diffusion fittings) see more details in the code.
- 07-10-30 ver9b
- For details, read the blog
- 08-03-25 ver 9c
- For details see the blog entry.
- 08-07-24 ver 9e
- Added two data set types for importing. Olympus .cvs file is now possible.
- 08-10-07 ver9f
- Added Excel file importing funciton (Four column, with Time-Frap-All Cell- Base, with the first line containg column name) and tab-delimited text file.
- 08/12/11 ver9g
- Replaced fitting function for double-exponential fitting, follwoing the recommendation of wavemetrics.
- Half-time estimation for double exponential fitting had a bug, when data range is below the half-max intensity. This is now done by extrapolation.
- 09-10-26 ver9h
- Fitted parameters appear in plot legend even when mobile fraction >1.0. (C. Tischer's suggestion)
- Added documentation for gammaQ=1 cases. See → 2.7. Evaluation of the Curve Fitting: Goodness of Fit (N. Goldsmith's suggestion)
- 10-03-03 ver9j
- Batch Processing funciton with averaging function (request from N. Goldsimth). See additional explanation in the documentation page
- K_FrapCalcCore() was modified so that averaged curve could be fitted as well. Since averaged curve does not have original normalizaton process is not required but then several parameters which are calculated during normalization will be missing. For this, flag “G_doAveraged” was added
- getAverageGApRatio(Gfilelist): Gap Ratio is calculated by averaging individual
- Fitting of averaged curve is done with actual standard deviation
- BatchFitting() was added to the last line of BatchAverageAndFit()
Export to OpenOffice- Tools:
Discussion
Hi-
This is an excellent procedure for Igor.
Quick request – is it possible to create an option for the Zeiss input with the following order:
Time, FRAP, Reference/Cell Part, Base/Background
(The model that I am using doesn't make us of of the outline of the cell- “allcell”)
And, a similar input for the batch.
Thank you.
Sincerely,
Elliott
Hi Elliott,
could you try the trial version below? If it works, please tell me so I could update the link above.
K_FRAPcalcV9k.ipf
thanks for also sending a mail to me. wiki should send me a automatic mail for notification but it didn't. I recently made a major renovation for this website so maybe something is wrong…
Sincerely,
Kota
Hi Kota,
This worked perfectly. Thank you for your time. I sincerely appreciate it. My lab mates are all impressed.
One more linked request, if possible.
Is it feasible to do a Batch import / Batch fit for the same Zeiss input order. (Time-Frap-Reference-Base).
Thank you again. Amazing.
Sincerely,
Elliott
Hi Elliot,
thanks for your feedback.
I updated again for the batch processing so please check if it works. I do not have your data so no debugging is done.
K_FRAPcalcV9k.ipf
if successful, you will have two types of results: (1) a table with fit parameters for individual curves, and (2) a plot of averaged curve. For the latter, curves are first averaged then the averaged curve is fitted. I recommend to use (1) and average Half max, mobile and immobile fraction, since some curves exhibit very low goodness of fit, and you better ignore them (if all are bad, then be doubtful with your model, that fitting equation is wrong). For this, you better check through the curves. After averaging parameters, you could easily generate a equation and plot it.
Sincerely,
Kota
Hi Kota,
Thanks so much. I really appreciate you giving it a try.
I just gave it a try. I had two error messages. The first read “aborted. ### wave index 1 have different time resolution compared to index 0 wave.”
The second message said “While executing WaveStats, the following error occured: expected wave name.”
Any thoughts?
I did have the table (1) above, but the graph did not come up.
Thanks.
Elliott
Hi Elliot,
thanks again for the feed back.
error #1 “aborted. ### wave index 1 have different time resolution compared to index 0 wave.”
this message tells you that the timings of acquisition were different in the data#1 compared to the data#0 ( List numbering is most likely in alphabetical order of the file name). When timings were various from experiment to experiment, it would not be possible to average data that you are feeding.
Two ways to deal with this are either: (1) Remove data#1 and try batch processing again, and if #0 and #2 have different time resolution, then remove # 2… and so on. If you know that timings were not controlled in experiments and various, then data averaging is impossible. Or (2) just average parameters listed in the table. This averaging should be done manually.
error #2 “While executing WaveStats, the following error occured: expected wave name. ”
this is probably because data averaging data was unsuccessful (error#1). I need to debug this, so it would be helpful if you could zip data files you have and send it to me.
Sincerely, Kota
Hi Kota,
Yes. I think that you are correct regarding the timing acquisition. I will go back and look at the raw data.
Thanks.
Elliott
Hi Elliot,
I updated the program again, so please check. The link is same as above, as I only replaced the file. For the second error, that was a real bug so I fixed it.
In your case, so called “gap ratio” becomes a serious issue since estimation of the degree of fluorescence loss due to FRAP bleaching is more difficult without all-cell fluorescence intensity. As I saw your data, there are only 5 time points before the FRAP bleach and this is not really enough for estimating the pre-bleach intensity.this has another effect, that the goodness of-fit becomes unreliable because of less quality of measurement error estimation. Please see the manual, as I updated some more explanation on this issue.
DataRequirements
2.7. Evaluation of the Curve Fitting: Goodness of Fit
Hi Kota,
I understand the issue regarding the gap ratio / pre-bleach time points.
I just tested the above link. I didn't get the same error message. It ran very smoothly.
Thank you again for your time. I look forward to continued use of the program.
Sincerely,
Elliott
What does FrapWidth mean in the Fit Panel? Does it mean the diameter or radius of the bleach area?
Thank you!
Aditya
Hi Aditya,
here is quote from my manual:
“In the “FRAP” panel, you should input the radius in “FRAP width” field. I did not use “FRAP radius” as the title of this field, since in case of Ellenberg fitting, strip-bleaching is assumed and size of such fitting does not correspond to “radius”. Half maximum of the recovery is estimated from the estimation curve generated by the equation (2-25).”
HTH, Kota
Thank you! I have one more.I use a Zeiss Confocal Setup for FRAP experiments. I usually get the results in a text file as Time-FRAP-Reference-Background…Does the option -4 columns: Time – FRAP – All Cell – Base
right? I mean does Base mean background data? Or alternatively should i ignore mt background data and choose the - 3 columns: Time – FRAP – Cell Part
option?
Thank you so much!
Aditya Iyer
Hi again,
“base” is the region outside where fluorescence is. If you are taking a time-lapse of a cell adhered to coverslip, then the base ROI should be taken from the area where no cell is existing. If that is your backgound, then that is the “base” (I mean “baseline” with this word). You should check where “backgound” in your data is coming from.
I cannot decide whether “all cell” or “cell part” should be used from the information you provided. Please describe what you are capturing, and where ROIs were made in your experiment…
Cheers, Kota
Hi,
Aditya
Hi, I am imaging Lipid Bilayers on Glass surface. I have to carry out FRAP measurements on these. I have three regions. One Reference, One FRAP region and the third which is background.
Aditya
If the “reference” is part of the lipid bilayer that is not FRAP-Bleached, then that will be considered as “cell part”. So in your case use
Import Data 4: Time - FRAP - CellPart - Base.
Please call me, maybe that would be better for discussing this.
Thank you so much for this!! I have a query however… After the fit, when i export the data, i do not find any data for the fitted curve or the residuals? Am i doing something wrong.?
Aditya
Hi Aditya,
there is no menu command for exporting them. To do manually, you could open Data Brawser (Data> Data Brawser) and look for the following waves.
1. fitted curves: there are two types, the fitted curve to the data points and the curve that was further corrected for acquisition bleaching. For both of these curves, name of the wave starts with “fit_” and added with experiment prefix (experiment name that you have given)
2. residuals are in the wave named with suffix “Res”
After finding them, you could create a table and export them using the native command such as Data>Save Waves > Save DlimitedText… and in the dialog window, check “From Target” (Target means the data table that you created with the fitted curve and residuals). Delimited file could be opened in general spreadsheet software.
HTH Kota