Please: Divide an Image with ImageJ

[Juan Francisco-2]

Dear all:
  please, I want to divide an image (TIFF, 16 bits) by its pixel value mean value (I get this figure on Analyse-->Measure). For that, I go to Proces--> Math--->Divide and so I can get the divided image. However I can see that ImageJ truncate the pixel values on the resulting image and gives the pixel value without decimals.
  Please, how its possible to get the pixel value on the divided image with 2 decimals at least?
  Thank you very much
  Juan Francisco

       
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[Michael Schmid]

On 8 May 2007, at 14:43, Juan Francisco wrote:
> (...)
>   Please, how its possible to get the pixel value on the divided  
> image with 2 decimals at least?


Hi Juan,

convert the image to float before dividing: Image>Type>32bit.

Michael

[Jonathan Hilmer]

Don't forget that you may wish to disable "Scale when converting"
prior to making it a 32bit.

Jonathan


On 5/8/07, Michael Schmid <[hidden email]> wrote:

> On 8 May 2007, at 14:43, Juan Francisco wrote:
> > (...)
> >   Please, how its possible to get the pixel value on the divided
> > image with 2 decimals at least?
>
>
> Hi Juan,
>
> convert the image to float before dividing: Image>Type>32bit.
>
> Michael

[Liu, Dongfang (NIH/NIAID) [F]]

Dear all,

Recently, I came across a problem with alexa568 and alexa647 on our TIRF system. I have to label my two proteins using the two different dyes (alexa568 and alexa647)simultaneously(because I have to use four dyes in one chamber, alxa488, alexa555, alexa568 and alexa647).

Problem: When I used 568nm laser to excite the alexa568 dyes on lipid bilayer(the emission filter is LP665), I got the fluorescence of alexa647 in alexa568 emission channel(because 568 laser can partially-10% excite the alexa567 laser.).

The protocol for acquiring image alexa568 and alexa647:
1, Acquire the alxa568 and alxa647 image in sequence.
2, Excitation laser for alexa568 is 568nm, emission for alexa568 is LP665
   Excitation laser for alexa568 is 647nm, emission for alexa647 is LP665

From the spectrum properties, we can know the fluorescence of Alexa647 can not breakthrough into alexa568 channel(even I used the same emission filter to acquiring the fluorescence of alexa568 and alexa647, because 647nm laser cannot excite the alexa568 dye)

What I want to do: I want to remove the fluorescence of alexa647 from alexa568 channel. How to do it? Any comments would be greatly appreciated!

Thank you very much! Have a nice day!

Best regards,

Don

[Wolfgang Schechinger]

Hi Don,

can you define a structure which is only labeled by one dye? Then you could measure the contribution of one dye to the crosstalk and determine the constant you have to multiply one image before subtracting it from the other. Maybe you have to make an experiment where you omit one of the dyes in question.

Just some thoughts,

Wo


-------- Original-Nachricht --------
Datum: Tue, 8 May 2007 12:29:36 -0400
Von: "Liu, Dongfang (NIH/NIAID) [F]" <[hidden email]>
An: [hidden email]
Betreff: Image correction help!

> Dear all,
>
> Recently, I came across a problem with alexa568 and alexa647 on our TIRF
> system. I have to label my two proteins using the two different dyes
> (alexa568 and alexa647)simultaneously(because I have to use four dyes in one
> chamber, alxa488, alexa555, alexa568 and alexa647).
>
> Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
> bilayer(the emission filter is LP665), I got the fluorescence of alexa647 in
> alexa568 emission channel(because 568 laser can partially-10% excite the
> alexa567 laser.).
>
> The protocol for acquiring image alexa568 and alexa647:
> 1, Acquire the alxa568 and alxa647 image in sequence.
> 2, Excitation laser for alexa568 is 568nm, emission for alexa568 is LP665
>    Excitation laser for alexa568 is 647nm, emission for alexa647 is LP665
>
> >From the spectrum properties, we can know the fluorescence of Alexa647
> can not breakthrough into alexa568 channel(even I used the same emission
> filter to acquiring the fluorescence of alexa568 and alexa647, because 647nm
> laser cannot excite the alexa568 dye)
>
> What I want to do: I want to remove the fluorescence of alexa647 from
> alexa568 channel. How to do it? Any comments would be greatly appreciated!
>
> Thank you very much! Have a nice day!
>
> Best regards,
>
> Don

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[Jacqueline Ross]

Dear Don,

Just a quick question regarding your image capture.

I'm just wondering why you are using an LP665 for emission collection of
your Alexa568.

You should have another more suitable barrier filter such as an OG590 or
something around 600nm.

If you are using an LP665, you will be missing out on a huge proportion
of your photons from the 568nm label.

Do you have any other filters in your system?

Cheers,

Jacqui.

Jacqueline Ross
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.health.auckland.ac.nz/biru/ 

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Liu, Dongfang (NIH/NIAID) [F]
Sent: 09 May 2007 04:30
To: [hidden email]
Subject: Image correction help!

Dear all,

Recently, I came across a problem with alexa568 and alexa647 on our TIRF
system. I have to label my two proteins using the two different dyes
(alexa568 and alexa647)simultaneously(because I have to use four dyes in
one chamber, alxa488, alexa555, alexa568 and alexa647).

Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
bilayer(the emission filter is LP665), I got the fluorescence of
alexa647 in alexa568 emission channel(because 568 laser can
partially-10% excite the alexa567 laser.).

The protocol for acquiring image alexa568 and alexa647:
1, Acquire the alxa568 and alxa647 image in sequence.
2, Excitation laser for alexa568 is 568nm, emission for alexa568 is
LP665
   Excitation laser for alexa568 is 647nm, emission for alexa647 is
LP665

From the spectrum properties, we can know the fluorescence of Alexa647
can not breakthrough into alexa568 channel(even I used the same emission
filter to acquiring the fluorescence of alexa568 and alexa647, because
647nm laser cannot excite the alexa568 dye)

What I want to do: I want to remove the fluorescence of alexa647 from
alexa568 channel. How to do it? Any comments would be greatly
appreciated!

Thank you very much! Have a nice day!

Best regards,

Don

[Liu, Dongfang (NIH/NIAID) [F]]

Dear Jacqui,

I greatly appreciate your help!
We are planning to buy a new filter for alexa568. Since you know the problem, could you give some suggestions about what kind of filter is good to distinguish the four dyes very well(alexa488, alexa555, alexa568 and alexa647). I need pretty narrow bandpasses filter to collect the peaking emission of each dye to calculate the correction factor. Can I use the spectral unmixing plugin to do this work?

Thank you very much!

Have a nice day!

Best regards,
Don

-----Original Message-----
From: Jacqui Ross [mailto:[hidden email]]
Sent: Tuesday, May 08, 2007 5:40 PM
To: List IMAGEJ
Subject: Re: Image correction help!

Dear Don,

Just a quick question regarding your image capture.

I'm just wondering why you are using an LP665 for emission collection of
your Alexa568.

You should have another more suitable barrier filter such as an OG590 or
something around 600nm.

If you are using an LP665, you will be missing out on a huge proportion
of your photons from the 568nm label.

Do you have any other filters in your system?

Cheers,

Jacqui.

Jacqueline Ross
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.health.auckland.ac.nz/biru/ 

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Liu, Dongfang (NIH/NIAID) [F]
Sent: 09 May 2007 04:30
To: [hidden email]
Subject: Image correction help!

Dear all,

Recently, I came across a problem with alexa568 and alexa647 on our TIRF
system. I have to label my two proteins using the two different dyes
(alexa568 and alexa647)simultaneously(because I have to use four dyes in
one chamber, alxa488, alexa555, alexa568 and alexa647).

Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
bilayer(the emission filter is LP665), I got the fluorescence of
alexa647 in alexa568 emission channel(because 568 laser can
partially-10% excite the alexa567 laser.).

The protocol for acquiring image alexa568 and alexa647:
1, Acquire the alxa568 and alxa647 image in sequence.
2, Excitation laser for alexa568 is 568nm, emission for alexa568 is
LP665
   Excitation laser for alexa568 is 647nm, emission for alexa647 is
LP665

From the spectrum properties, we can know the fluorescence of Alexa647
can not breakthrough into alexa568 channel(even I used the same emission
filter to acquiring the fluorescence of alexa568 and alexa647, because
647nm laser cannot excite the alexa568 dye)

What I want to do: I want to remove the fluorescence of alexa647 from
alexa568 channel. How to do it? Any comments would be greatly
appreciated!

Thank you very much! Have a nice day!

Best regards,

Don

[Jacqueline Ross]

Hi Don,

I'm happy to give some suggestions on filters. If you can give me some
more information on your system, that would make things easier.

Please tell me what laser(s) you have and what microscope. I was
assuming that you had an ArKr laser with 488, 568, 647 lines but it
would be helpful if you can let me know for sure. What laser line are
you using to excite the Alexa555? What emission filters do you have
already?

Your plan may require using unmixing because the fluorophores you plan
to use (Alexa 488, 555, 568, 647) are rather close.

In fact, I would think that the 568/647 separation will be less of a
problem than trying to separate out the Alexa555. The combination of 555
and 568 is not ideal.

I assume that you have used the spectrum viewer from Molecular Probes at
http://probes.invitrogen.com/resources/spectraviewer/ (or elsewhere) to
check all your overlaps and excitation problems.

A lot depends on the intensity of the different labels.

We may need to take this discussion about filters off-list since it
doesn't really relate directly to ImageJ unless others are also
interested?

I can't help you with the spectral unmixing plugin because I have not
used it. However, with our Leica system, you still need to make sure
that you have adequate controls to use their unmixing algorithm.

I'm sure that all these algorithms are similar in what is required with
regard to controls. There are probably others on the ImageJ list that
can respond better with regard to the specifics of the ImageJ plugin.

Kind regards,

Jacqui

Jacqueline Ross
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.health.auckland.ac.nz/biru/ 

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Liu, Dongfang (NIH/NIAID) [F]
Sent: 10 May 2007 01:41
To: [hidden email]
Subject: Re: Image correction help!

Dear Jacqui,

I greatly appreciate your help!
We are planning to buy a new filter for alexa568. Since you know the
problem, could you give some suggestions about what kind of filter is
good to distinguish the four dyes very well(alexa488, alexa555, alexa568
and alexa647). I need pretty narrow bandpasses filter to collect the
peaking emission of each dye to calculate the correction factor. Can I
use the spectral unmixing plugin to do this work?

Thank you very much!

Have a nice day!

Best regards,
Don

-----Original Message-----
From: Jacqui Ross [mailto:[hidden email]]
Sent: Tuesday, May 08, 2007 5:40 PM
To: List IMAGEJ
Subject: Re: Image correction help!

Dear Don,

Just a quick question regarding your image capture.

I'm just wondering why you are using an LP665 for emission collection of
your Alexa568.

You should have another more suitable barrier filter such as an OG590 or
something around 600nm.

If you are using an LP665, you will be missing out on a huge proportion
of your photons from the 568nm label.

Do you have any other filters in your system?

Cheers,

Jacqui.

Jacqueline Ross
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.health.auckland.ac.nz/biru/ 

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
Liu, Dongfang (NIH/NIAID) [F]
Sent: 09 May 2007 04:30
To: [hidden email]
Subject: Image correction help!

Dear all,

Recently, I came across a problem with alexa568 and alexa647 on our TIRF
system. I have to label my two proteins using the two different dyes
(alexa568 and alexa647)simultaneously(because I have to use four dyes in
one chamber, alxa488, alexa555, alexa568 and alexa647).

Problem: When I used 568nm laser to excite the alexa568 dyes on lipid
bilayer(the emission filter is LP665), I got the fluorescence of
alexa647 in alexa568 emission channel(because 568 laser can
partially-10% excite the alexa567 laser.).

The protocol for acquiring image alexa568 and alexa647:
1, Acquire the alxa568 and alxa647 image in sequence.
2, Excitation laser for alexa568 is 568nm, emission for alexa568 is
LP665
   Excitation laser for alexa568 is 647nm, emission for alexa647 is
LP665

From the spectrum properties, we can know the fluorescence of Alexa647
can not breakthrough into alexa568 channel(even I used the same emission
filter to acquiring the fluorescence of alexa568 and alexa647, because
647nm laser cannot excite the alexa568 dye)

What I want to do: I want to remove the fluorescence of alexa647 from
alexa568 channel. How to do it? Any comments would be greatly
appreciated!

Thank you very much! Have a nice day!

Best regards,

Don