Confocal Microscopy List

Frap analysis

Classic

List

Threaded

8 messages Options

Anchall ............

Frap analysis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello all,
I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA USING
IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
someone can tell me step by step procedure to analyse frap data .I use leica
sp5 for collecting frap data.
Hope to hear from you all soon
Thanks
Anchal

Kevin Braeckmans

Re: Frap analysis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Anchal,

It would be helpful if you could give some more details - what are you
trying to measure, what kind of sample are you using, do you want to obtain
quantitative data and if so, which FRAP method would you like to use etc.

Very generally, the difficulty is not the image processing, which usually is
just averaging the fluorescence intensity in some ROI and normalizing that
to a reference area and the fluorescence before bleaching. If you want to
obtain quantitative data, the real challenge is to perform the experiment
according to the limits implied in the FRAP model (which are not always
clearly discussed in articles). Only then one can expect to obtain
reasonable values from a best fit of the model to the recovery curve
(obtaining a value does not necessarily mean it is a correct one).

If you are starting from scratch, I would advise the following:
1. Define your experiment, the sample geometry being the most important
factor (3D extended sample, sample with limited thickness, sample of limited
volume)
2. Choose a FRAP method which is suitable for your experiment and which can
be performed by your instrument.
3. Practice on some reference solutions of known diffusion coefficient to
see if the method works correctly (each FRAP method has some limits which
are implied in the mathematical derivation - make sure to perform your
experiment accordingly)
4. Apply the method to your actual samples.

You also might want to check out the archives since this topic has been
discussed several times on this list.

Best regards,

Kevin

Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Anchal Chandra
> Verzonden: donderdag 24 januari 2008 18:31
> Aan: [hidden email]
> Onderwerp: Frap analysis
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello all,
> I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA
> USING
> IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
> someone can tell me step by step procedure to analyse frap data .I use
> leica
> sp5 for collecting frap data.
> Hope to hear from you all soon
> Thanks
> Anchal

Anchall ............

Re: Frap analysis

In reply to this post by Anchall ............

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Kevin,
Thanks a lot for replying.I work with different RAS proteins
which are mostly localised in plasma membrane and some intracellular
organelles like golgi and ER.All these proteins have c terminal post
translational modifications .Now what I want to see is if different
post translational mutants of these proteins have similar or different
kinetics inside living cells.I am using MDCK cells for these expt.I
want to frap the mutants and the wt and see if there is any difference
in the diffusion kinetics and to find out if these modifications have
any role to play in defining localisation and kinetics of these
proteins.I will like to quantify the data once I get these frap
mesurements. Regarding which frap model I should use I am bit
confused.I dont know which model I should use to fit my data.
Also I didnt understand how sample geometry can help me select the
frap method .You also mentioned about checking some reference
solutions first. Can you guide me what kind of samples I should use
for reference.
Hope you will reply soon
Regards
Anchal

On Jan 25, 2008 7:47 AM, Kevin Braeckmans <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Anchal,
>
> It would be helpful if you could give some more details - what are you
> trying to measure, what kind of sample are you using, do you want to obtain
> quantitative data and if so, which FRAP method would you like to use etc.
>
> Very generally, the difficulty is not the image processing, which usually is
> just averaging the fluorescence intensity in some ROI and normalizing that
> to a reference area and the fluorescence before bleaching. If you want to
> obtain quantitative data, the real challenge is to perform the experiment
> according to the limits implied in the FRAP model (which are not always
> clearly discussed in articles). Only then one can expect to obtain
> reasonable values from a best fit of the model to the recovery curve
> (obtaining a value does not necessarily mean it is a correct one).
>
> If you are starting from scratch, I would advise the following:
> 1. Define your experiment, the sample geometry being the most important
> factor (3D extended sample, sample with limited thickness, sample of limited
> volume)
> 2. Choose a FRAP method which is suitable for your experiment and which can
> be performed by your instrument.
> 3. Practice on some reference solutions of known diffusion coefficient to
> see if the method works correctly (each FRAP method has some limits which
> are implied in the mathematical derivation - make sure to perform your
> experiment accordingly)
> 4. Apply the method to your actual samples.
>
> You also might want to check out the archives since this topic has been
> discussed several times on this list.
>
> Best regards,
>
> Kevin
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Harelbekestraat 72
> 9000 Ghent
> Belgium
> Tel: +32 (0)9 264.80.78
> Fax: +32 (0)9 264.81.89
> > -----Oorspronkelijk bericht-----
> > Van: Confocal Microscopy List [mailto:[hidden email]]
> > Namens Anchal Chandra
> > Verzonden: donderdag 24 januari 2008 18:31
> > Aan: [hidden email]
> > Onderwerp: Frap analysis
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> >
> > Hello all,
> > I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA
> > USING
> > IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
> > someone can tell me step by step procedure to analyse frap data .I use
> > leica
> > sp5 for collecting frap data.
> > Hope to hear from you all soon
> > Thanks
> > Anchal
>

Stewart, Angela

Remove from list

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Please remove me from this list.

Thank you,

Angie Stewart
PerkinElmer Life and Analytical Sciences
Life Science Instrument Sales
Mobile: 561-309-1013
Office: 561-745-9177
Email: [hidden email]
www.perkinelmer.com
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Anchall ............
Sent: Friday, January 25, 2008 4:38 AM
To: [hidden email]
Subject: Re: Frap analysis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Kevin,
Thanks a lot for replying.I work with different RAS proteins
which are mostly localised in plasma membrane and some intracellular
organelles like golgi and ER.All these proteins have c terminal post
translational modifications .Now what I want to see is if different
post translational mutants of these proteins have similar or different
kinetics inside living cells.I am using MDCK cells for these expt.I
want to frap the mutants and the wt and see if there is any difference
in the diffusion kinetics and to find out if these modifications have
any role to play in defining localisation and kinetics of these
proteins.I will like to quantify the data once I get these frap
mesurements. Regarding which frap model I should use I am bit
confused.I dont know which model I should use to fit my data.
Also I didnt understand how sample geometry can help me select the
frap method .You also mentioned about checking some reference
solutions first. Can you guide me what kind of samples I should use
for reference.
Hope you will reply soon
Regards
Anchal

On Jan 25, 2008 7:47 AM, Kevin Braeckmans <[hidden email]>
wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Anchal,
>
> It would be helpful if you could give some more details - what are you
> trying to measure, what kind of sample are you using, do you want to
obtain
> quantitative data and if so, which FRAP method would you like to use
etc.
>
> Very generally, the difficulty is not the image processing, which
usually is
> just averaging the fluorescence intensity in some ROI and normalizing
that
> to a reference area and the fluorescence before bleaching. If you want
to
> obtain quantitative data, the real challenge is to perform the
experiment
> according to the limits implied in the FRAP model (which are not
always
> clearly discussed in articles). Only then one can expect to obtain
> reasonable values from a best fit of the model to the recovery curve
> (obtaining a value does not necessarily mean it is a correct one).
>
> If you are starting from scratch, I would advise the following:
> 1. Define your experiment, the sample geometry being the most
important
> factor (3D extended sample, sample with limited thickness, sample of
limited
> volume)
> 2. Choose a FRAP method which is suitable for your experiment and
which can
> be performed by your instrument.
> 3. Practice on some reference solutions of known diffusion coefficient
to
> see if the method works correctly (each FRAP method has some limits
which
> are implied in the mathematical derivation - make sure to perform your
> experiment accordingly)
> 4. Apply the method to your actual samples.
>
> You also might want to check out the archives since this topic has
been

> discussed several times on this list.
>
> Best regards,
>
> Kevin
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Harelbekestraat 72
> 9000 Ghent
> Belgium
> Tel: +32 (0)9 264.80.78
> Fax: +32 (0)9 264.81.89
> > -----Oorspronkelijk bericht-----
> > Van: Confocal Microscopy List [mailto:[hidden email]]
> > Namens Anchal Chandra
> > Verzonden: donderdag 24 januari 2008 18:31
> > Aan: [hidden email]
> > Onderwerp: Frap analysis
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> >
> > Hello all,
> > I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA
> > USING
> > IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice

if
> > someone can tell me step by step procedure to analyse frap data .I
use
> > leica
> > sp5 for collecting frap data.
> > Hope to hear from you all soon
> > Thanks
> > Anchal
>

Julio Vazquez

Re: Frap analysis

In reply to this post by Anchall ............

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Anchal,

Perhaps you could have a look at the review articles referenced below, and see among the references if there are any that deal with work similar to what you plan to do; thwe first two, you should be able to look up in PubMed and download for free.

Carrero G, McDonald D, Crawford C, et al. Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29: 14-28 (2003).

Lippincott-Schwartz J, Altan_Bonnet N, Patterson G. Photobleaching and photoactivation: following protein dynamics in living cells. Nature Reviews S7-S14 (2003) (check Medline for exact reference)

and this Chapter in Live Cell Imaging: A Laboratory Manual. CSHL Press, Goldman and Spector (Eds):

G Rabut and J Ellenberg: Photobleaching Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells: FRAP, iFRAP, and FLIP. Ch 7, pp 101-126. Lots of useful references there...

That could be a start...

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,

Seattle, WA 98109-1024

www.fhcrc.org

Macilvain, Terrence

Hi Angie!: Remove from list

In reply to this post by Stewart, Angela

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Angie! Our VoX Rocks!! We made many at Vandy smile this past week!!

You may need to remind Scott to edit his seminar presentation when he
lands in FL. Scott planned to enhance the Vandy presentation for you and
may forget due to his 100+ e-mails per day and ~20 voice mail messages
every day.

Good luck with your events this week!
See you in 8 days!
Terry

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Stewart, Angela
Sent: Friday, January 25, 2008 8:46 AM
To: [hidden email]
Subject: Remove from list

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Please remove me from this list.

Thank you,

Angie Stewart
PerkinElmer Life and Analytical Sciences
Life Science Instrument Sales
Mobile: 561-309-1013
Office: 561-745-9177
Email: [hidden email]
www.perkinelmer.com
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Anchall ............
Sent: Friday, January 25, 2008 4:38 AM
To: [hidden email]
Subject: Re: Frap analysis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Kevin,
Thanks a lot for replying.I work with different RAS proteins
which are mostly localised in plasma membrane and some intracellular
organelles like golgi and ER.All these proteins have c terminal post
translational modifications .Now what I want to see is if different
post translational mutants of these proteins have similar or different
kinetics inside living cells.I am using MDCK cells for these expt.I
want to frap the mutants and the wt and see if there is any difference
in the diffusion kinetics and to find out if these modifications have
any role to play in defining localisation and kinetics of these
proteins.I will like to quantify the data once I get these frap
mesurements. Regarding which frap model I should use I am bit
confused.I dont know which model I should use to fit my data.
Also I didnt understand how sample geometry can help me select the
frap method .You also mentioned about checking some reference
solutions first. Can you guide me what kind of samples I should use
for reference.
Hope you will reply soon
Regards
Anchal

On Jan 25, 2008 7:47 AM, Kevin Braeckmans <[hidden email]>
wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Anchal,
>
> It would be helpful if you could give some more details - what are you
> trying to measure, what kind of sample are you using, do you want to
obtain
> quantitative data and if so, which FRAP method would you like to use
etc.
>
> Very generally, the difficulty is not the image processing, which
usually is
> just averaging the fluorescence intensity in some ROI and normalizing
that
> to a reference area and the fluorescence before bleaching. If you want
to
> obtain quantitative data, the real challenge is to perform the
experiment
> according to the limits implied in the FRAP model (which are not
always
> clearly discussed in articles). Only then one can expect to obtain
> reasonable values from a best fit of the model to the recovery curve
> (obtaining a value does not necessarily mean it is a correct one).
>
> If you are starting from scratch, I would advise the following:
> 1. Define your experiment, the sample geometry being the most
important
> factor (3D extended sample, sample with limited thickness, sample of
limited
> volume)
> 2. Choose a FRAP method which is suitable for your experiment and
which can
> be performed by your instrument.
> 3. Practice on some reference solutions of known diffusion coefficient
to
> see if the method works correctly (each FRAP method has some limits
which
> are implied in the mathematical derivation - make sure to perform your
> experiment accordingly)
> 4. Apply the method to your actual samples.
>
> You also might want to check out the archives since this topic has
been

if
> > someone can tell me step by step procedure to analyse frap data .I
use
> > leica
> > sp5 for collecting frap data.
> > Hope to hear from you all soon
> > Thanks
> > Anchal
>

jens rietdorf

Re: Frap analysis

In reply to this post by Anchall ............

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Anchal,

Kota Miura from CMCI @ EMBL Heidelberg has written IgorPro routines for FRAP
analysis.
http://www.embl.org/cmci/downloads/frap_analysis.html
I remember it can use Leica input. Best contact Kota directly and ask him.

Cheers, jens

Anchal Chandra wrote:

>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello all,
> I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA USING
> IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
> someone can tell me step by step procedure to analyse frap data .I use
> leica
> sp5 for collecting frap data.
> Hope to hear from you all soon
> Thanks
> Anchal
>
>

--
View this message in context: http://www.nabble.com/Frap-analysis-tp15073261p15171003.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

xavier Sanjuan

Re: Frap analysis

In reply to this post by Anchall ............

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Perhaps you will also find helpful the following:
http://www.embl-heidelberg.de/eamnet/html/teaching_modules.html

Regards,

Xavi.

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]]En
nombre de Jens Rietdorf
Enviado el: dimecres, 30 / gener / 2008 09:19
Para: [hidden email]
Asunto: Re: Frap analysis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Anchal,

Kota Miura from CMCI @ EMBL Heidelberg has written IgorPro routines for FRAP
analysis.
http://www.embl.org/cmci/downloads/frap_analysis.html
I remember it can use Leica input. Best contact Kota directly and ask him.

Cheers, jens

Anchal Chandra wrote:

--
View this message in context:
http://www.nabble.com/Frap-analysis-tp15073261p15171003.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.