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Total fluorescent intensity within area

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Mike Tighe

Total fluorescent intensity within area

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Using Fiji/ImageJ I would like to define an area within an image and compare the total fluorescent intensity for a particular channel and compare it to another defined area within the the same image. Is there a simple way to do this?

Thanks for any help!

Mike

Chris Tully

Re: Total fluorescent intensity within area

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Mike,

I do not use ImageJ all that often, however, since I am working on a brand
new computer I went and downloaded the latest version of ImageJ to look at
your problem. First On the Analyze menu, look for the Set Measurements...
command. This displays a dialog where you can select the measurements to
be taken. By default, ImageJ measures Area, Min/Max and Mean
intensity/density. To do what you have described, simply check off the
Integrated Density measurement (in addition to or in place of the defaults)
and click OK. Then draw the area to be analyzed using one of the ROI tools
(rectangle, ellipse, freehand; buttons below the menu bar). Finally choose
Analyze|Measure to measure the first area. Then drag the ROI to the second
area and choose Analyze|Measure again. The Results window will
automatically be displayed the first time you click Analyze|Measure.

Chris

Chris Tully
Microscopy and Image Analysis Expert
[hidden email]
240-475-9753 (c)

[image: View my profile on LinkedIn]<http://www.linkedin.com/in/christully/>

On Wed, Feb 27, 2013 at 11:14 AM, Mike Tighe <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Using Fiji/ImageJ I would like to define an area within an image and
> compare the total fluorescent intensity for a particular channel and
> compare it to another defined area within the the same image. Is there a
> simple way to do this?
>
>
>
> Thanks for any help!
>
> Mike
>

Matthew Nicholas-2

Re: Total fluorescent intensity within area

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Mike,
Just to add a small tip on to what Chris described -- the measurement
function can be performed by pressing M on the keyboard, which allows you
to easily move around the image and make measurements without needing any
menus.

Incidentally, in case you have not considered it, you may need to be
cautious when comparing integrated intensities (for example, if the
background is different in the two regions, this will contribute to the
measured difference).

Matt

--
Matthew Nicholas
Medical Scientist Training Program Student
Laboratory of Arne Gennerich
Department of Anatomy and Structural Biology
Albert Einstein College of Medicine
Forchheimer Building, Room 628
1300 Morris Park Avenue
Bronx, New York 10461
718.430.3446
[hidden email]

On Wed, Feb 27, 2013 at 11:36 AM, Chris Tully <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Mike,
>
> I do not use ImageJ all that often, however, since I am working on a brand
> new computer I went and downloaded the latest version of ImageJ to look at
> your problem. First On the Analyze menu, look for the Set Measurements...
> command. This displays a dialog where you can select the measurements to
> be taken. By default, ImageJ measures Area, Min/Max and Mean
> intensity/density. To do what you have described, simply check off the
> Integrated Density measurement (in addition to or in place of the defaults)
> and click OK. Then draw the area to be analyzed using one of the ROI tools
> (rectangle, ellipse, freehand; buttons below the menu bar). Finally choose
> Analyze|Measure to measure the first area. Then drag the ROI to the second
> area and choose Analyze|Measure again. The Results window will
> automatically be displayed the first time you click Analyze|Measure.
>
>
> Chris
>
> Chris Tully
> Microscopy and Image Analysis Expert
> [hidden email]
> 240-475-9753 (c)
>
> [image: View my profile on LinkedIn]<
> http://www.linkedin.com/in/christully/>
>
>
> On Wed, Feb 27, 2013 at 11:14 AM, Mike Tighe <[hidden email]
> >wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Using Fiji/ImageJ I would like to define an area within an image and
> > compare the total fluorescent intensity for a particular channel and
> > compare it to another defined area within the the same image. Is there a
> > simple way to do this?
> >
> >
> >
> > Thanks for any help!
> >
> > Mike
> >
>

<[hidden email]>

Mike Tighe

Re: Total fluorescent intensity within area

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

OK... so I am able to measure Integrated density/Area/raw Intensity in both channel one and two for a given area (Thanks!!). What I am trying to do in the end is to measure multiple areas with in an Image and between images and show that the T cells (channel 1) are more likely to be in the Bcell (channel 2) in group A vs. group B. So.....

If I were to measure Bcell area based on channel 2 and look at Total (integrated density or Raw integrated density??) of channel 1 within the same area can I divide the total Intensity and divide by the total area selected within an image? What would that give me?

What does the Integrated Intensity and Raw Integrated represent?

Thanks again for your help!
Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Matthew Nicholas [[hidden email]]
Sent: Wednesday, February 27, 2013 12:00 PM
To: [hidden email]
Subject: Re: Total fluorescent intensity within area

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Mike,
Just to add a small tip on to what Chris described -- the measurement
function can be performed by pressing M on the keyboard, which allows you
to easily move around the image and make measurements without needing any
menus.

Incidentally, in case you have not considered it, you may need to be
cautious when comparing integrated intensities (for example, if the
background is different in the two regions, this will contribute to the
measured difference).

Matt

--
Matthew Nicholas
Medical Scientist Training Program Student
Laboratory of Arne Gennerich
Department of Anatomy and Structural Biology
Albert Einstein College of Medicine
Forchheimer Building, Room 628
1300 Morris Park Avenue
Bronx, New York 10461
718.430.3446
[hidden email]

On Wed, Feb 27, 2013 at 11:36 AM, Chris Tully <[hidden email]> wrote:

<[hidden email]>

Julio Vazquez

Re: Total fluorescent intensity within area

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Raw integrated density is the sum of all intensities of all pixels. If image is uncalibrated (distance unit is pixel), Integrated density and Raw integrated density are the same. If image is calibrated in real units, Raw Integrated Intensity is the same as above, while Integrated density is normalized to one area unit, I believe. Total intensity divided by number of pixels equals mean intensity per pixel (which you can also obtain from imagej, just select that parameter in the Set Measurements tab). If you divide by the calibrated area, then you get the mean intensity per area unit.

To analyze several regons across different images, use the ROI manager tool (Edit > Selection > Add to manager).

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org/

==

On Feb 28, 2013, at 8:05 AM, Mike Tighe wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> OK... so I am able to measure Integrated density/Area/raw Intensity in both channel one and two for a given area (Thanks!!). What I am trying to do in the end is to measure multiple areas with in an Image and between images and show that the T cells (channel 1) are more likely to be in the Bcell (channel 2) in group A vs. group B. So.....
>
> If I were to measure Bcell area based on channel 2 and look at Total (integrated density or Raw integrated density??) of channel 1 within the same area can I divide the total Intensity and divide by the total area selected within an image? What would that give me?
>
> What does the Integrated Intensity and Raw Integrated represent?
>
> Thanks again for your help!
> Mike
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on behalf of Matthew Nicholas [[hidden email]]
> Sent: Wednesday, February 27, 2013 12:00 PM
> To: [hidden email]
> Subject: Re: Total fluorescent intensity within area
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mike,
> Just to add a small tip on to what Chris described -- the measurement
> function can be performed by pressing M on the keyboard, which allows you
> to easily move around the image and make measurements without needing any
> menus.
>
> Incidentally, in case you have not considered it, you may need to be
> cautious when comparing integrated intensities (for example, if the
> background is different in the two regions, this will contribute to the
> measured difference).
>
> Matt
>
> --
> Matthew Nicholas
> Medical Scientist Training Program Student
> Laboratory of Arne Gennerich
> Department of Anatomy and Structural Biology
> Albert Einstein College of Medicine
> Forchheimer Building, Room 628
> 1300 Morris Park Avenue
> Bronx, New York 10461
> 718.430.3446
> [hidden email]
>
> On Wed, Feb 27, 2013 at 11:36 AM, Chris Tully <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Mike,
>>
>> I do not use ImageJ all that often, however, since I am working on a brand
>> new computer I went and downloaded the latest version of ImageJ to look at
>> your problem. First On the Analyze menu, look for the Set Measurements...
>> command. This displays a dialog where you can select the measurements to
>> be taken. By default, ImageJ measures Area, Min/Max and Mean
>> intensity/density. To do what you have described, simply check off the
>> Integrated Density measurement (in addition to or in place of the defaults)
>> and click OK. Then draw the area to be analyzed using one of the ROI tools
>> (rectangle, ellipse, freehand; buttons below the menu bar). Finally choose
>> Analyze|Measure to measure the first area. Then drag the ROI to the second
>> area and choose Analyze|Measure again. The Results window will
>> automatically be displayed the first time you click Analyze|Measure.
>>
>>
>> Chris
>>
>> Chris Tully
>> Microscopy and Image Analysis Expert
>> [hidden email]
>> 240-475-9753 (c)
>>
>> [image: View my profile on LinkedIn]<
>> http://www.linkedin.com/in/christully/>
>>
>>
>> On Wed, Feb 27, 2013 at 11:14 AM, Mike Tighe <[hidden email]
>>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Using Fiji/ImageJ I would like to define an area within an image and
>>> compare the total fluorescent intensity for a particular channel and
>>> compare it to another defined area within the the same image. Is there a
>>> simple way to do this?
>>>
>>>
>>>
>>> Thanks for any help!
>>>
>>> Mike
>>>
>>
>
>
>
> <[hidden email]>