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Re: 2D area calculations

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Patel, Ankit S

Re: 2D area calculations

Hi everyone, I am new to ImageJ. I need some help, could someone please help me with following,

I have images from cardiac muscle with arteriols and empty space in between. I want to calculate area of each of these three.

Thank you.
Sincerely,
Ankit

"Being happy doesn't mean that everything is perfect. It means that you've decided to look beyond the imperfections."

Francesco Lassandro

Re: 2D area calculations

you can try yawi 2D available at http://yawi3d.sourceforge.net/

francesco

----- Original Message -----
From: "Patel, Ankit S" <[hidden email]>
To: <[hidden email]>
Sent: Monday, April 09, 2007 8:13 PM
Subject: Re: 2D area calculations

Hi everyone, I am new to ImageJ. I need some help, could someone please help
me with following,

I have images from cardiac muscle with arteriols and empty space in between.
I want to calculate area of each of these three.

Thank you.
Sincerely,
Ankit

"Being happy doesn't mean that everything is perfect. It means that you've
decided to look beyond the imperfections."

Patel, Ankit S

Re: 2D area calculations

I couldn't find software for windows

Thank you for your reply.

"Being happy doesn't mean that everything is perfect. It means that you've decided to look beyond the imperfections."

________________________________

From: ImageJ Interest Group on behalf of Francesco Lassandro
Sent: Mon 4/9/2007 4:08 PM
To: [hidden email]
Subject: Re: 2D area calculations

you can try yawi 2D available at http://yawi3d.sourceforge.net/

francesco

----- Original Message -----
From: "Patel, Ankit S" <[hidden email]>
To: <[hidden email]>
Sent: Monday, April 09, 2007 8:13 PM
Subject: Re: 2D area calculations

Hi everyone, I am new to ImageJ. I need some help, could someone please help
me with following,

I have images from cardiac muscle with arteriols and empty space in between.
I want to calculate area of each of these three.

Thank you.
Sincerely,
Ankit

"Being happy doesn't mean that everything is perfect. It means that you've
decided to look beyond the imperfections."

Wolfgang Schechinger

Densitometry of Western Blots

In reply to this post by Patel, Ankit S

Dear ImageJ Experts,

I want to quantitate signals on X-Ray films obtained by Western Blot / ECL.
Actually, I just scanned the films with an ordinary flatbed scanner
(transparency mode, internally 10 bit), that gives me 8bit tiff
images. I use the Ctrl_1/2/3 sequence from ImageJ to generate density
graphs of each lane which then are integrated with the wand tool.

Two issues now make me think and I am looking for a good solution:

1) Strong signals yield spots on the film which probably are bigger
than the actual spot was on the gel. Longer exposure times make larger spots)

2) I am worried about the meaning of the area under the curve data as
I have scanned a microplate of a colorimetric assay and compared the
OD values obtained by the photometer with the grey values of the
wells obtained by scanning. The relationship is not linear unless I
would plot the log of the grey values against the OD. That only would
make sense if one applied Lamber-Beers's law I/Io=10^(-ecd) on the
data where I is the grey value and ecd is the OD value from the photometer.

I also read in a tutorial on a laser densitometer from Molecular
Devices that this machine would convert the densitometric values to
greyscales for storage using a logarithmic relation that would
resemble the equation mentioned above.

My goal is to compare the amounts of proteins on the blots. May I
assume (within all the limits the use of xray films has) that double
the amount of protein yields double the area under the curve or is
the reality (even under the constraints mentioned) more complex. Is
there a reason (or even necessity) to use the log of the area in
order to obtain correct values?

Thanks for your help!

Wo

Francesco Lassandro

Re: 2D area calculations

In reply to this post by Patel, Ankit S

Ankit, I use this ImageJ plugin on windows. you should download the zip
file, unzip the file in the folder plugin of ImageJ and then compile and run
it from the ImageJ menu.

bye
Francesco

----- Original Message -----
From: "Patel, Ankit S" <[hidden email]>
To: <[hidden email]>
Sent: Tuesday, April 10, 2007 5:49 AM
Subject: Re: 2D area calculations

I couldn't find software for windows

Thank you for your reply.

"Being happy doesn't mean that everything is perfect. It means that you've
decided to look beyond the imperfections."

________________________________

From: ImageJ Interest Group on behalf of Francesco Lassandro
Sent: Mon 4/9/2007 4:08 PM
To: [hidden email]
Subject: Re: 2D area calculations

you can try yawi 2D available at http://yawi3d.sourceforge.net/

francesco

----- Original Message -----
From: "Patel, Ankit S" <[hidden email]>
To: <[hidden email]>
Sent: Monday, April 09, 2007 8:13 PM
Subject: Re: 2D area calculations

Hi everyone, I am new to ImageJ. I need some help, could someone please help
me with following,

I have images from cardiac muscle with arteriols and empty space in between.
I want to calculate area of each of these three.

Thank you.
Sincerely,
Ankit

"Being happy doesn't mean that everything is perfect. It means that you've
decided to look beyond the imperfections."

Wayne Rasband

Re: Densitometry of Western Blots

In reply to this post by Wolfgang Schechinger

The best solution is to scan the film along with a calibrated OD step
tablet and to generate a calibration curve using the procedure
described at

http://rsb.info.nih.gov/ij/docs/examples/calibration/

Search the Web for EK1523422 or EK1523406 to find companies that sell
Kodak step tablets.

-wayne

On Apr 10, 2007, at 6:14 PM, Wolfgang Schechinger wrote:

> Dear ImageJ Experts,
>
> I want to quantitate signals on X-Ray films obtained by Western Blot /
> ECL.
> Actually, I just scanned the films with an ordinary flatbed scanner
> (transparency mode, internally 10 bit), that gives me 8bit tiff
> images. I use the Ctrl_1/2/3 sequence from ImageJ to generate density
> graphs of each lane which then are integrated with the wand tool.
>
> Two issues now make me think and I am looking for a good solution:
>
> 1) Strong signals yield spots on the film which probably are bigger
> than the actual spot was on the gel. Longer exposure times make larger
> spots)
>
> 2) I am worried about the meaning of the area under the curve data as
> I have scanned a microplate of a colorimetric assay and compared the
> OD values obtained by the photometer with the grey values of the wells
> obtained by scanning. The relationship is not linear unless I would
> plot the log of the grey values against the OD. That only would make
> sense if one applied Lamber-Beers's law I/Io=10^(-ecd) on the data
> where I is the grey value and ecd is the OD value from the photometer.
>
> I also read in a tutorial on a laser densitometer from Molecular
> Devices that this machine would convert the densitometric values to
> greyscales for storage using a logarithmic relation that would
> resemble the equation mentioned above.
>
> My goal is to compare the amounts of proteins on the blots. May I
> assume (within all the limits the use of xray films has) that double
> the amount of protein yields double the area under the curve or is the
> reality (even under the constraints mentioned) more complex. Is there
> a reason (or even necessity) to use the log of the area in order to
> obtain correct values?
>
> Thanks for your help!
>
> Wo
>

Jonathan Hilmer

Re: Densitometry of Western Blots

Wayne,

Would you happen to know what kind of certification the Kodak/Tiffen
targets have? I've seen that version from Stouffer has a substantial
price difference between the ones that are just calibrated and the
ones which are both calibrated and certified: 40 vs 110 USD.

Jonathan

On 4/11/07, Wayne Rasband <[hidden email]> wrote:

> The best solution is to scan the film along with a calibrated OD step
> tablet and to generate a calibration curve using the procedure
> described at
>
> http://rsb.info.nih.gov/ij/docs/examples/calibration/
>
> Search the Web for EK1523422 or EK1523406 to find companies that sell
> Kodak step tablets.
>
> -wayne

Damien Haton-Pietrin

Re: Densitometry of Western Blots

------------------------------------------------------------------------
>It is possible to calibrate manually to draw first a black dot with
>paintbrush tool and second to measure a white area therefore setting
>measurement ... And calibrating with strait line.
>Or tacking a white area (as zero) on the scanning blot and and black dot
>drawwing on scanned frame (100) subsequently setting measurment with
>calibration toolbox.
>An other technic largely used in our laboratory is to draw a line on total
>length of a band and measure it to show the level of gray values.
>What do you think about?

>Dr. Damien PIETRIN
>Laboratoire de Neurobiologie et Transgénèse
>UPRES 3143
>bâtiment Montéclair Génopôle Ouest
>4, Rue Larrey
>49033 CHU Angers
>tel: 02-41-35-36-37
>Poste 42598
____________________________________________________________________________
_

> Wayne,
>
> Would you happen to know what kind of certification the Kodak/Tiffen
> targets have? I've seen that version from Stouffer has a substantial
> price difference between the ones that are just calibrated and the
> ones which are both calibrated and certified: 40 vs 110 USD.
>
> Jonathan
>
>
>
> On 4/11/07, Wayne Rasband <[hidden email]> wrote:
>> The best solution is to scan the film along with a calibrated OD step
>> tablet and to generate a calibration curve using the procedure
>> described at
>>
>> http://rsb.info.nih.gov/ij/docs/examples/calibration/
>>
>> Search the Web for EK1523422 or EK1523406 to find companies that sell
>> Kodak step tablets.
>>
>> -wayne

Le 11/04/07 20:44, « Jonathan Hilmer » <[hidden email]> a écrit :

Gabriel Landini

Re: Densitometry of Western Blots

On Wednesday 11 April 2007 21:53:15 Damien PIETRIN wrote:
> ------------------------------------------------------------------------
> >It is possible to calibrate manually to draw first a black dot with
> >paintbrush tool and second to measure a white area therefore setting
> >measurement ... And calibrating with strait line.

Erm... no.

> >Or tacking a white area (as zero) on the scanning blot and and black dot
> >drawwing on scanned frame (100) subsequently setting measurment with
> >calibration toolbox.

What are you calibrating against? If you use drawn spots, you are inventing
the calibration.

> >An other technic largely used in our laboratory is to draw a line on
> > total length of a band and measure it to show the level of gray values.

That sounds like a profile of the grey levels. What is the relation betwewn
grey 10 and grey 100? That is answered in a porperly calibrated image.
Measuring the grey levels only (with an unknown sensor response) is no
calibration at all.
A peak 3 times darker than another may mean completely different things
depending on the curve response of the sensor.
None of the methods above shed any light on this problem. Wayne's solution
will give you an idea of the relations between the scanned greyvalues of the
stepping calibration strips.

G.