FRAP analysis

[Colin Rickman-3]

Hi

I'm looking for software to analyse FRAP experiments - both intensity measurement and the subsequent model based curve fitting. Obviously the intensity part is relatively simple in ImageJ/FIJI. Doe anybody have a plugin for the subsequent analysis? I've been using the frap_analysis Matlab program for measurement/fitting but the fitting is based on a 2D system.

Any help gratefully received.

Regards

Colin

Dr Colin Rickman
Life Science Interface
Department of Chemistry (WP 2.03)
School of Engineering and Physical Sciences
Heriot-Watt University
Edinburgh
EH14 4AS

Tel: [phone] (Office)

http://www.lifescienceinterface.hw.ac.uk
http://www.eps.hw.ac.uk/departments/chemistry/cr.htm



--
Heriot-Watt University is a Scottish charity
registered under charity number SC000278.

[Unruh, Jay-2]

Colin,

Fitting the data to a rising exponential is fairly easy in Origin or many other programs (you can actually even do it in excel if you want--just define the chi squared and use the solver add-in to minimize it).  It sounds like you are more interested in a geometry based analysis (2D, 3D, reaction-diffusion, etc).  The only thing I have seen on this is the Virtual Cell project and their FRAP tool, though it is more designed for model comparison than actual data fitting.  Of course, there are several other system-specific things out there, but those don't really help you unless you are working on the exact same system.  Unfortunately, beyond simple exponential fitting, FRAP analysis usually involves complex numerical differential equation solutions that aren't typically available in non system-specific programs.

Jay

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Colin Rickman
Sent: Wednesday, September 14, 2011 7:34 AM
To: [hidden email]
Subject: FRAP analysis

Hi

I'm looking for software to analyse FRAP experiments - both intensity measurement and the subsequent model based curve fitting. Obviously the intensity part is relatively simple in ImageJ/FIJI. Doe anybody have a plugin for the subsequent analysis? I've been using the frap_analysis Matlab program for measurement/fitting but the fitting is based on a 2D system.

Any help gratefully received.

Regards

Colin

Dr Colin Rickman
Life Science Interface
Department of Chemistry (WP 2.03)
School of Engineering and Physical Sciences Heriot-Watt University Edinburgh
EH14 4AS

Tel: [phone] (Office)

http://www.lifescienceinterface.hw.ac.uk
http://www.eps.hw.ac.uk/departments/chemistry/cr.htm



--
Heriot-Watt University is a Scottish charity registered under charity number SC000278.

[Daniel James White]

Hi Colin,

My colleagues who work on FRAP analysis (cc'd here) tell me that the usual approach by measuring in in a fixed area is less than ideal,
as it takes no account of the way the bleaching "spreads" out of the area the laser hit.

see
http://www.sciencedirect.com/science/article/pii/S0006349510010283

If you are really serious about taking care of the details and getting a more meaningful result...
then this approach should be helpful.
It is also in 2D.... but one could imagine a 3D implementation that takes account of the shape of the PSF of the bleaching light.

They have it implemented in matlab.... but we could make a Fiji plugin to do  the same method.
http://publications.mpi-cbg.de/getDocument.html?id=ff8080812a729fa4012a7f6324040007

If you or anyone has resources to port this to an imageJ/Fiji plugin,
then I would be happy to help out with communication and design/implementation etc.
But the hard core maths part is over my head...probably.

cheers

Dan



On Sep 15, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:

> Date:    Wed, 14 Sep 2011 13:33:53 +0100
> From:    Colin Rickman <[hidden email]>
> Subject: FRAP analysis
>
> Hi
>
> I'm looking for software to analyse FRAP experiments - both intensity measurement and the subsequent model based curve fitting. Obviously the intensity part is relatively simple in ImageJ/FIJI. Doe anybody have a plugin for the subsequent analysis? I've been using the frap_analysis Matlab program for measurement/fitting but the fitting is based on a 2D system.
>
> Any help gratefully received.
>
> Regards
>
> Colin
>
> Dr Colin Rickman
> Life Science Interface
> Department of Chemistry (WP 2.03)
> School of Engineering and Physical Sciences
> Heriot-Watt University
> Edinburgh
> EH14 4AS
>
> Tel: [phone] (Office)
>
> http://www.lifescienceinterface.hw.ac.uk
> http://www.eps.hw.ac.uk/departments/chemistry/cr.htm

Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[ayo raheem]

.
I have an IHC stained section i want to be semi-quantified using Image J that a  colleague suggested and have no idea at all on how this can be done. i will like someone to take me through this, having brown and blue as my positive and negative colours respectively in the stained.
Thanks
KAYBE

[Vitali Khvatkov-2]

Assuming you are talking about ImmunoHistoChemistry staining, there is
imageJ plugin for color deconvolution that may work:
http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html

It may be included in Fiji distribution of ImageJ, in which case i would
recommend to use Fiji. http://fiji.sc/wiki/index.php/Fiji

In general, trying to segment IHC staining just on color, without morphology
or structure detectors is not very reliable method.

Just in case you are willing to try not just ImageJ, there are other
free software options available.

- Ilastik http://www.ilastik.org/ can do "point and click" adaptive object
selection and segmentation (no measurement)
- Simagis Live Web Pathology http://web-pathology.net/ , Web Application
that can process whole IHC slides for Histology stain deconvolution and
measurement in the Cloud , no need for software. It can take common slide
scanner formats like Aiperio etc.  (Free up to 2GB limit)

Both options above have integration with ImageJ

Regards,
Vitali


On Thu, Sep 15, 2011 at 2:35 PM, ayo raheem <[hidden email]> wrote:

>
>
> .
> I have an IHC stained section i want to be semi-quantified using Image J
> that a  colleague suggested and have no idea at all on how this can be done.
> i will like someone to take me through this, having brown and blue as my
> positive and negative colours respectively in the stained.
> Thanks
> KAYBE
>
>

--
Vitali Khvatkov
Smart Imaging Technologies Co.
[phone]
www.simagis.com

[Gabriel Landini]

On Friday 16 Sep 2011, Vitali Khvatkov wrote:
> Assuming you are talking about ImmunoHistoChemistry staining, there is
> imageJ plugin for color deconvolution that may work:
> http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html

Sorry to keep repeating the same thing over and over, but obviously not
everybody reads the "note in red" in the colour deconvolution page.

Just to repeat it once again, you cannot *quantify* IHC staining because there
is no simple linear or robust relation between the intensity of the stain
given by the chromogen in the result, and the *amount of antigen* (which is
what one would like to quantify).
The intensity of the stain depends on a number of parameters that the user
cannot control because there is a chain of amplification steps which are just
not controllable AND at least DAB does not follow the Beer-Lambert law..

It is quite remarkable that for a long time this was well known, but more
recently, perhaps due to availability of easier/affordable imaging programs,
more and more people insist in taking this kind of approach.

IHC are more of a "yes/no" answer; you detect an antigen or you do not, rather
than "exactly how much" depending on intensity.

There are unfortunately some papers in the literature that have somehow made
it through the less-than-perfect referee system of some journals.
Obviously those authors (and the referees) show that they do not understand
what stoichiometry means and what the Beer-Lambert law is.

Colour devonvolution is useful for segmentation, but not for quantification of
gene expression or antigen quantification.

Cheers

Gabriel

[Jacqueline Ross]

Hi Kaybe,

It sounds like you are using something similar to DAB and haematoxylin. Try using the Colour Deconvolution plugin. It's available here: http://www.dentistry.bham.ac.uk/landinig/software/software.html

Kind regards,

Jacqui.



Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of ayo raheem
Sent: Friday, 16 September 2011 7:35 a.m.
To: [hidden email]
Subject: IHC QUANTIFCATION WITH IMAGE J



.
I have an IHC stained section i want to be semi-quantified using Image J that a  colleague suggested and have no idea at all on how this can be done. i will like someone to take me through this, having brown and blue as my positive and negative colours respectively in the stained.
Thanks
KAYBE

[Vilppu Tuominen-2]

Please have a look at our open source IHC analysis software: ImmunoRatio (for ER, PR, and Ki-67) and ImmunoMembrane (for HER2). They both come as free web applications as well as ImageJ plugins. The public web versions have already been used to analyze over 20 000 images and are routinely applied in clinical diagnostics (laboratory-specific calibration is advisable prior to routine usage).

http://jvsmicroscope.uta.fi/immunoratio/
http://jvsmicroscope.uta.fi/immunoratio-plugin/ 

http://jvsmicroscope.uta.fi/immunomembrane/
http://jvsmicroscope.uta.fi/immunomembrane-plugin/

Both software are based on the excellent color deconvolution plugin provided by prof. Landini.  And, as prof. Landini explained in his earlier post, quantifying IHC is somewhat problematic. However, visual assessment of IHC stain intensity has been a routine diagnostic procedure for a long time. For example, the ASCO/CAP criteria for HER2 assessment in breast cancer are widely applied world-wide and they are based (partly) on the stain intensity assessment. So, even though there are fundamental issues with the quantitation, it has been proved to correlate with patient prognosis. With regards to automated HER2 IHC analysis software, we are just merely trying to mimic the manual (visual) assessment, and for these kinds of applications, color deconvolution provides a good approximation.

As for our software, ImmunoRatio uses color deconvolution only for segmentation and ImmunoMembrane to roughly classify samples into three categories (according to ASCO/CAP criteria; user-calibratable).

Best regards,
Vilppu Tuominen

--
Vilppu Tuominen, MSc

Cancer Biology research group
Institute of Biosciences and Medical Technology
University of Tampere
33014 Tampere, Finland

(mail) [hidden email]
(gsm)  [phone]
(tel)  [phone]
(fax)  [phone]

[Gabriel Landini]

On Monday 19 Sep 2011 13:05:53 Vilppu Tuominen wrote:
> quantifying IHC is somewhat problematic. However, visual assessment
> of IHC stain intensity has been a routine diagnostic procedure for a long
> time.

Hi Vilppu,
Sure, it has been used for a long time, but that does not make it any more
quantifiable.
Likewise, "nuclear hyperchromatism" on H&E sections has been used for many
years as an indirect indication of hyperploidy in tumour cells, yet neither
man nor machine can quantify this for the same reason that haematoxylin is not
a stoichiometric stain... we know that it cannot be measured reliably but it
is still mentioned in pathology reports.
Feulgen stain, on the other hand, is stoichiometric, yet paradoxically no-one
uses Feulgen stain for diagnosis perhaps because it is seen as more
complicated as it is has one (!) hydrolysis step that needs controlling (and
how many steps in IHC?).

> For example, the ASCO/CAP criteria for HER2 assessment in breast
> cancer are widely applied world-wide and they are based (partly) on the
> stain intensity assessment.  So, even though there are fundamental issues
> with the quantitation, it has been proved to correlate with patient
> prognosis.

You mean "scoring" (like: nothing, a bit, some, a lot) but that is not truly
quantitation either. There is no numerical factor that relates these ("a lot"
is not n times "a bit").

It is understandable that in the desperate search for meaningful prognostic
markers this ended up being used. But the problem remains, and moving to the
next step to just use the optical density of the stain as marker, will produce
numbers that are not precise quantities representing what we want to know.

We should not forget that HER2 assessment remains as "scores" and did not
evolve into quantifying the intensity. The reason is precisely that it is not
possible to control all the amplification steps in the staining procedure to
obtain reliable information on the quantity of antigen present.

Kind regards

Gabriel

[Vitali Khvatkov-2]

Gabriel is absolutely correct, our studies of the subject lead to the same
conclusions.

Color vector and its deconvolution from RGB image can be used for
thresholding  and further  characterization of features (dimensional,
morphological, spatial etc.), but not direct measurement of expression.

There are some other ways that arguably may work for measuring expression
(against baseline), but they involve multi-spectral imaging and composite
analysis of multiple spectral layers with filters of specific bandwidth.
Cameras and filters exist (e.g. VariSpec filters from Caliper LifeSciences).
But this story is probably outside of the scope of this post..

Vitali Khvatkov
Smart Imaging Technologies Co.
www.live.simagis.com



-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Gabriel Landini
Sent: Saturday, September 17, 2011 4:52 AM
To: [hidden email]
Subject: Re: IHC QUANTIFCATION WITH IMAGE J

On Friday 16 Sep 2011, Vitali Khvatkov wrote:
> Assuming you are talking about ImmunoHistoChemistry staining, there is
> imageJ plugin for color deconvolution that may work:
> http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html

Sorry to keep repeating the same thing over and over, but obviously not
everybody reads the "note in red" in the colour deconvolution page.

Just to repeat it once again, you cannot *quantify* IHC staining because
there is no simple linear or robust relation between the intensity of the
stain given by the chromogen in the result, and the *amount of antigen*
(which is what one would like to quantify).
The intensity of the stain depends on a number of parameters that the user
cannot control because there is a chain of amplification steps which are
just not controllable AND at least DAB does not follow the Beer-Lambert
law..

It is quite remarkable that for a long time this was well known, but more
recently, perhaps due to availability of easier/affordable imaging programs,
more and more people insist in taking this kind of approach.

IHC are more of a "yes/no" answer; you detect an antigen or you do not,
rather than "exactly how much" depending on intensity.

There are unfortunately some papers in the literature that have somehow made
it through the less-than-perfect referee system of some journals.
Obviously those authors (and the referees) show that they do not understand
what stoichiometry means and what the Beer-Lambert law is.

Colour devonvolution is useful for segmentation, but not for quantification
of gene expression or antigen quantification.

Cheers

Gabriel

[Vilppu Tuominen-2]

I fully agree with Gabriel that using the stain intensity alone as a
prognostic marker is just plainly wrong. And it is indeed puzzling to
see many people doing this despite the facts.

With regards to automated HER2 scoring, the terminology is a bit fuzzy.
I agree that 'scoring' does not count as 'quantitation', but in order to
approximate visual scoring, our analysis software (ImmunoMembrane) has
to internally quantify the stain intensity prior to classification--and
even shows this raw data to the user for calibration purposes. So, in
this case, perhaps a more accurate description would be 'semi-quantitative'.

All the best,
Vilppu

--
Vilppu Tuominen, MSc

Cancer Biology research group
Institute of Biosciences and Medical Technology
University of Tampere
33014 Tampere, Finland

(mail) [hidden email]
(gsm)  [phone]
(tel)  [phone]
(fax)  [phone]

On 20.9.2011 7:01, IMAGEJ automatic digest system wrote:

> On Monday 19 Sep 2011 13:05:53 Vilppu Tuominen wrote:
>> quantifying IHC is somewhat problematic. However, visual assessment
>> of IHC stain intensity has been a routine diagnostic procedure for a long
>> time.
 >

> Hi Vilppu,
> Sure, it has been used for a long time, but that does not make it any more
> quantifiable.
> Likewise, "nuclear hyperchromatism" on H&E sections has been used for many
> years as an indirect indication of hyperploidy in tumour cells, yet neither
> man nor machine can quantify this for the same reason that haematoxylin is not
> a stoichiometric stain... we know that it cannot be measured reliably but it
> is still mentioned in pathology reports.
> Feulgen stain, on the other hand, is stoichiometric, yet paradoxically no-one
> uses Feulgen stain for diagnosis perhaps because it is seen as more
> complicated as it is has one (!) hydrolysis step that needs controlling (and
> how many steps in IHC?).
>
>> For example, the ASCO/CAP criteria for HER2 assessment in breast
>> cancer are widely applied world-wide and they are based (partly) on the
>> stain intensity assessment.  So, even though there are fundamental issues
>> with the quantitation, it has been proved to correlate with patient
>> prognosis.

 >

> You mean "scoring" (like: nothing, a bit, some, a lot) but that is not truly
> quantitation either. There is no numerical factor that relates these ("a lot"
> is not n times "a bit").
>
> It is understandable that in the desperate search for meaningful prognostic
> markers this ended up being used. But the problem remains, and moving to the
> next step to just use the optical density of the stain as marker, will produce
> numbers that are not precise quantities representing what we want to know.
>
> We should not forget that HER2 assessment remains as "scores" and did not
> evolve into quantifying the intensity. The reason is precisely that it is not
> possible to control all the amplification steps in the staining procedure to
> obtain reliable information on the quantity of antigen present.
>
> Kind regards
>
> Gabriel

[Jacqueline Ross]

Hi All,

Just a quick note regarding the recent discussion.

I recommended using the Colour Deconvolution plugin, which I use a lot and find very useful especially for DAB (immunostaining = brown) and Haematoxylin (=blue) separation, since Haematoxylin is commonly used as a counterstain but then makes the segmentation of the immuno difficult with standard RGB splitting.

However, I assumed (perhaps wrongly!) that the original enquirer (Kaybe) was measuring areas (or number of features/cells stained) rather than being interested in intensity differences.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Vilppu Tuominen
Sent: Wednesday, 21 September 2011 7:56 p.m.
To: [hidden email]
Subject: Re: IHC QUANTIFCATION WITH IMAGE J

I fully agree with Gabriel that using the stain intensity alone as a
prognostic marker is just plainly wrong. And it is indeed puzzling to
see many people doing this despite the facts.

With regards to automated HER2 scoring, the terminology is a bit fuzzy.
I agree that 'scoring' does not count as 'quantitation', but in order to
approximate visual scoring, our analysis software (ImmunoMembrane) has
to internally quantify the stain intensity prior to classification--and
even shows this raw data to the user for calibration purposes. So, in
this case, perhaps a more accurate description would be 'semi-quantitative'.

All the best,
Vilppu

--
Vilppu Tuominen, MSc

Cancer Biology research group
Institute of Biosciences and Medical Technology
University of Tampere
33014 Tampere, Finland

(mail) [hidden email]
(gsm)  [phone]
(tel)  [phone]
(fax)  [phone]

On 20.9.2011 7:01, IMAGEJ automatic digest system wrote:

> On Monday 19 Sep 2011 13:05:53 Vilppu Tuominen wrote:
>> quantifying IHC is somewhat problematic. However, visual assessment
>> of IHC stain intensity has been a routine diagnostic procedure for a long
>> time.
 >

> Hi Vilppu,
> Sure, it has been used for a long time, but that does not make it any more
> quantifiable.
> Likewise, "nuclear hyperchromatism" on H&E sections has been used for many
> years as an indirect indication of hyperploidy in tumour cells, yet neither
> man nor machine can quantify this for the same reason that haematoxylin is not
> a stoichiometric stain... we know that it cannot be measured reliably but it
> is still mentioned in pathology reports.
> Feulgen stain, on the other hand, is stoichiometric, yet paradoxically no-one
> uses Feulgen stain for diagnosis perhaps because it is seen as more
> complicated as it is has one (!) hydrolysis step that needs controlling (and
> how many steps in IHC?).
>
>> For example, the ASCO/CAP criteria for HER2 assessment in breast
>> cancer are widely applied world-wide and they are based (partly) on the
>> stain intensity assessment.  So, even though there are fundamental issues
>> with the quantitation, it has been proved to correlate with patient
>> prognosis.

 >

> You mean "scoring" (like: nothing, a bit, some, a lot) but that is not truly
> quantitation either. There is no numerical factor that relates these ("a lot"
> is not n times "a bit").
>
> It is understandable that in the desperate search for meaningful prognostic
> markers this ended up being used. But the problem remains, and moving to the
> next step to just use the optical density of the stain as marker, will produce
> numbers that are not precise quantities representing what we want to know.
>
> We should not forget that HER2 assessment remains as "scores" and did not
> evolve into quantifying the intensity. The reason is precisely that it is not
> possible to control all the amplification steps in the staining procedure to
> obtain reliable information on the quantity of antigen present.
>
> Kind regards
>
> Gabriel

[Vitali Khvatkov-2]

We have adopted ImmunoRatio solution for Simagis Live servers and tested it
on avaialbe library of Ki-67 markers, comparing results with prior analysis.
The results are practically usable although, as expected, setting
adjustments are required for images from different sources to account for
process variability. Here are App screen shots:
http://portal.sliderocket.com/AXFYY/ImmunoRatio-with-SImagis-Live

The App now is freely available to all Simagis Live users. Due to constants
in underlying ImageJ code, this App only works on single images and not the
whole slide like native Simagis Live Apps.

Regards,

Vitali Khvatkov
713-589-3500
live.simagis.com


-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Jacqui
Ross
Sent: Wednesday, September 21, 2011 4:47 PM
To: [hidden email]
Subject: Re: IHC QUANTIFCATION WITH IMAGE J

Hi All,

Just a quick note regarding the recent discussion.

I recommended using the Colour Deconvolution plugin, which I use a lot and
find very useful especially for DAB (immunostaining = brown) and
Haematoxylin (=blue) separation, since Haematoxylin is commonly used as a
counterstain but then makes the segmentation of the immuno difficult with
standard RGB splitting.

However, I assumed (perhaps wrongly!) that the original enquirer (Kaybe) was
measuring areas (or number of features/cells stained) rather than being
interested in intensity differences.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: [phone] Ext 87438
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Vilppu
Tuominen
Sent: Wednesday, 21 September 2011 7:56 p.m.
To: [hidden email]
Subject: Re: IHC QUANTIFCATION WITH IMAGE J

I fully agree with Gabriel that using the stain intensity alone as a
prognostic marker is just plainly wrong. And it is indeed puzzling to see
many people doing this despite the facts.

With regards to automated HER2 scoring, the terminology is a bit fuzzy.
I agree that 'scoring' does not count as 'quantitation', but in order to
approximate visual scoring, our analysis software (ImmunoMembrane) has to
internally quantify the stain intensity prior to classification--and even
shows this raw data to the user for calibration purposes. So, in this case,
perhaps a more accurate description would be 'semi-quantitative'.

All the best,
Vilppu

--
Vilppu Tuominen, MSc

Cancer Biology research group
Institute of Biosciences and Medical Technology University of Tampere
33014 Tampere, Finland

(mail) [hidden email]
(gsm)  [phone]
(tel)  [phone]
(fax)  [phone]

On 20.9.2011 7:01, IMAGEJ automatic digest system wrote:

> On Monday 19 Sep 2011 13:05:53 Vilppu Tuominen wrote:
>> quantifying IHC is somewhat problematic. However, visual assessment
>> of IHC stain intensity has been a routine diagnostic procedure for a
>> long time.
 >

> Hi Vilppu,
> Sure, it has been used for a long time, but that does not make it any
> more quantifiable.
> Likewise, "nuclear hyperchromatism" on H&E sections has been used for
> many years as an indirect indication of hyperploidy in tumour cells,
> yet neither man nor machine can quantify this for the same reason that
> haematoxylin is not a stoichiometric stain... we know that it cannot
> be measured reliably but it is still mentioned in pathology reports.
> Feulgen stain, on the other hand, is stoichiometric, yet paradoxically
> no-one uses Feulgen stain for diagnosis perhaps because it is seen as
> more complicated as it is has one (!) hydrolysis step that needs
> controlling (and how many steps in IHC?).
>
>> For example, the ASCO/CAP criteria for HER2 assessment in breast
>> cancer are widely applied world-wide and they are based (partly) on
>> the stain intensity assessment.  So, even though there are
>> fundamental issues with the quantitation, it has been proved to
>> correlate with patient prognosis.

 >

> You mean "scoring" (like: nothing, a bit, some, a lot) but that is not
> truly quantitation either. There is no numerical factor that relates these
> ("a lot"
> is not n times "a bit").
>
> It is understandable that in the desperate search for meaningful
> prognostic markers this ended up being used. But the problem remains,
> and moving to the next step to just use the optical density of the
> stain as marker, will produce numbers that are not precise quantities
> representing what we want to know.
>
> We should not forget that HER2 assessment remains as "scores" and did
> not evolve into quantifying the intensity. The reason is precisely
> that it is not possible to control all the amplification steps in the
> staining procedure to obtain reliable information on the quantity of
> antigen present.
>
> Kind regards
>
> Gabriel

[Herbie-6]

Dear IJ-macro experts,

a list created by

        List.set( "Preferred Mac", "Mini" );
            .
            .
            .
        List.set( "Apple Campus Street No.", "1" );

and stored by

        setMetadata( "Info", List.getList() );

as meta-data of a tiff image, leads to an unordered display of the
list when the image's meta-data is recalled by "Show Info..."

Is there a way to influence the display order of the list key/value-pairs?

Best

                   Herbie

          ------------------------
          <http://www.gluender.de>

[dscho]

Hi Gluender,

On Thu, 29 Sep 2011, Gluender wrote:

> a list created by
>
> List.set( "Preferred Mac", "Mini" );
>            .
>            .
>            .
> List.set( "Apple Campus Street No.", "1" );
>
> and stored by
>
> setMetadata( "Info", List.getList() );
>
> as meta-data of a tiff image, leads to an unordered display of the list
> when the image's meta-data is recalled by "Show Info..."

As far as I can see, List.set() operates on a java.util.Properties
instance, which subclasses the Hashtable (which "orders" the keys in a
combination of insertion order and the keys' hash).

> Is there a way to influence the display order of the list
> key/value-pairs?

Unfortunately, there is no macro function (yet?) to extract the keys
(which you could then sort by Arrays.sort() and construct the string
List.getList() would give you yourself).

But you can always resort to javascript by using the eval("script", ...)
macro function.

Ciao,
Johannes

[Rasband, Wayne (NIH/NIMH) [E]]

On Sep 29, 2011, at 3:54 PM, Gluender wrote:

> Dear IJ-macro experts,
>
> a list created by
>
> List.set( "Preferred Mac", "Mini" );
>            .
>            .
>            .
> List.set( "Apple Campus Street No.", "1" );
>
> and stored by
>
> setMetadata( "Info", List.getList() );
>
> as meta-data of a tiff image, leads to an unordered display of the
> list when the image's meta-data is recalled by "Show Info..."
>
> Is there a way to influence the display order of the list key/value-pairs?

In the ImageJ 1.45q daily build, the List.getList() macro function returns the key/value pairs in alphanumeric order.

-wayne

[Herbie-6]

>On Sep 29, 2011, at 3:54 PM, Gluender wrote:
>
>>  Dear IJ-macro experts,
>>
>>  a list created by
>>
>> List.set( "Preferred Mac", "Mini" );
>>             .
>>             .
>>             .
>> List.set( "Apple Campus Street No.", "1" );
>>
>>  and stored by
>>
>> setMetadata( "Info", List.getList() );
>>
>>  as meta-data of a tiff image, leads to an unordered display of the
>>  list when the image's meta-data is recalled by "Show Info..."
>>
>>  Is there a way to influence the display order of the list key/value-pairs?
>
>In the ImageJ 1.45q daily build, the List.getList() macro function
>returns the key/value pairs in alphanumeric order.
>
>-wayne

Many thanks for the hints to Johannes Schindelin.
I suspected hashing to be the case...

Enormous thanks for the again surprisingly quick reaction to Wayne.
Ordering is of great help if lists are not only searched but
presented to human readers. Great achievement!

Thanks for IJ in general and for the helpful community of this list.

Best

                   Herbie

          ------------------------
          <http://www.gluender.de>