Immunofluorescence and quantifying the intensity

[sean_incali]

Hello everyone

I need to do some immunofluorescence and would like to quantify the intensity in ROIs.

I've done similar tasks using older version of ImageJ, but I remember having an awful time with background subtraction. (it was an issue with different levels of intensities in areas chosen for background subtraction)

My questions are,

1. What will be a proper way to background subtract if I'm using a confocal microscope(not LSM)?  Should I have a blank coverslips and take a picture to use as a background?

2. Does rolling ball algorithm also work for immunofluorescence pictures?

3. What image type is best for quantifying the intensities in a ROIs when performing immunofluorescence? (Tiff, BMP etc)

4. If I need to split the images in to different channels(say red and green), what levels of gray is best? 8 bit 16, or 32?  Can ImageJ analyze particle (for quantifying gray intensity) images that are 16 or 32 bit?

Thanks for any info in advance.

Sean





     

[Johannes-P. Koch]

Dear Sean,


1) I suppose the best background would be a coverslips with your sample on
it and just a secondary antibody (not your primary....). You should do this
anyway as a control for your experimental setup (detecting unspecific
binding of 2nd ab), thus you can use this for background measurements. You
should acquire your images always with the same settings in laser intensity,
detector gain, amplifier offset and so on...so it might be better to test
these on your "normal" slides and then go over to measure the background.
Another strategy might be to take a ROI in your images that does not show
any significant fluorescence. Both approaches have cons and pros.

2) Why not?

3) The broader the dynamic range of your image, the better....so what is
available on your scope?? Maybe 12-bit? Do store uncompressed files, maybe
raw files (if available!?) I suggest TIFF

4) a) RGB is 3x8-bit...(without an alpha channel, of course)....check here
http://rsb.info.nih.gov/ij/docs/menus/image.html#type
    b) I expect you to be capable of testing this yourself....it is one
simple click to check whether it is possible or not...

Best,
Johannes


----- Original Message -----
From: "sean k" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, June 03, 2009 12:03 AM
Subject: Immunofluorescence and quantifying the intensity

> Hello everyone
>
> I need to do some immunofluorescence and would like to quantify the
> intensity in ROIs.
>
> I've done similar tasks using older version of ImageJ, but I remember
> having an awful time with background subtraction. (it was an issue with
> different levels of intensities in areas chosen for background
> subtraction)
>
> My questions are,
>
> 1. What will be a proper way to background subtract if I'm using a
> confocal microscope(not LSM)?  Should I have a blank coverslips and take a
> picture to use as a background?
>
> 2. Does rolling ball algorithm also work for immunofluorescence pictures?
>
> 3. What image type is best for quantifying the intensities in a ROIs when
> performing immunofluorescence? (Tiff, BMP etc)
>
> 4. If I need to split the images in to different channels(say red and
> green), what levels of gray is best? 8 bit 16, or 32?  Can ImageJ analyze
> particle (for quantifying gray intensity) images that are 16 or 32 bit?
>
> Thanks for any info in advance.
>
> Sean
>
>
>
>
>
>
>
>

--
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[Joel Sheffield]

Let me add to this excellent summary that you also have to make sure:

a.  That you are accounting for photobleaching during exposure,
focussing, etc.  For many fluorphores, significant bleaching can
occur within minutes.  If your examination time varies for different
samples, you may have some problems with accuracy.

b.  Make sure that your images are taken below saturation of the
fluorphores.

Joel

> Dear Sean,
>
>
> 1) I suppose the best background would be a coverslips with your sample on
> it and just a secondary antibody (not your primary....). You should do this
> anyway as a control for your experimental setup (detecting unspecific
> binding of 2nd ab), thus you can use this for background measurements. You
> should acquire your images always with the same settings in laser intensity,
> detector gain, amplifier offset and so on...so it might be better to test
> these on your "normal" slides and then go over to measure the background.
> Another strategy might be to take a ROI in your images that does not show
> any significant fluorescence. Both approaches have cons and pros.
>
> 2) Why not?
>
> 3) The broader the dynamic range of your image, the better....so what is
> available on your scope?? Maybe 12-bit? Do store uncompressed files, maybe
> raw files (if available!?) I suggest TIFF
>
> 4) a) RGB is 3x8-bit...(without an alpha channel, of course)....check here
> http://rsb.info.nih.gov/ij/docs/menus/image.html#type
>     b) I expect you to be capable of testing this yourself....it is one
> simple click to check whether it is possible or not...
>
> Best,
> Johannes
>
>
> ----- Original Message -----
> From: "sean k" <[hidden email]>
> To: <[hidden email]>
> Sent: Wednesday, June 03, 2009 12:03 AM
> Subject: Immunofluorescence and quantifying the intensity
>
>
> > Hello everyone
> >
> > I need to do some immunofluorescence and would like to quantify the
> > intensity in ROIs.
> >
> > I've done similar tasks using older version of ImageJ, but I remember
> > having an awful time with background subtraction. (it was an issue with
> > different levels of intensities in areas chosen for background
> > subtraction)
> >
> > My questions are,
> >
> > 1. What will be a proper way to background subtract if I'm using a
> > confocal microscope(not LSM)?  Should I have a blank coverslips and take a
> > picture to use as a background?
> >
> > 2. Does rolling ball algorithm also work for immunofluorescence pictures?
> >
> > 3. What image type is best for quantifying the intensities in a ROIs when
> > performing immunofluorescence? (Tiff, BMP etc)
> >
> > 4. If I need to split the images in to different channels(say red and
> > green), what levels of gray is best? 8 bit 16, or 32?  Can ImageJ analyze
> > particle (for quantifying gray intensity) images that are 16 or 32 bit?
> >
> > Thanks for any info in advance.
> >
> > Sean
> >
> >
> >
> >
> >
> >
> >
> >
>
>
> --
> Ich verwende die kostenlose Version von SPAMfighter für private Anwender,
> die bei mir bis jetzt 224 Spammails entfernt hat.
> Rund 6 Millionen Leute nutzen SPAMfighter schon.
> Laden Sie SPAMfighter kostenlos herunter: http://www.spamfighter.com/lde

--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]
[phone], fax [phone]
http://astro.temple.edu/~jbs