Excluding black (255) from an image

[elambeth]

I am working with red stained images of frog liver.  There are pigment cells in the liver that are black, and should not count for the quantification of the red stain.  I want to know the mean gray value of the red pixels, and I know this is on the histogram, but I do not know how to exclude the black pigment cells from the histogram (basically I want a histogram of the red stain only).  Is there any way to exclude the black?

Emily Lambeth, Graduate Student
Department of Biological Sciences
Notre Dame University

[Theresa Swayne]

Hi Emily,

If I understand the problem correctly, one way to solve it is:

-- (Image>Adjust>Threshold. Set upper threshold to 254. Lower  
threshold should be 0. Select "Over/Under" from the drop-down menu to  
see the excluded pixels in green.
-- Analyze > Set Measurements. Check "Mean" and "Limit to Threshold."
-- Analyze > Measure.

This will give you the mean of the pixels that are between 0 and 254,  
inclusive.

Hope this helps.
Theresa

On Aug 11, 2008, at 1:59 PM, elambeth wrote:

> I am working with red stained images of frog liver.  There are  
> pigment cells
> in the liver that are black, and should not count for the  
> quantification of
> the red stain.  I want to know the mean gray value of the red  
> pixels, and I
> know this is on the histogram, but I do not know how to exclude the  
> black
> pigment cells from the histogram (basically I want a histogram of  
> the red
> stain only).  Is there any way to exclude the black?
>

------------------------------------
Theresa C. Swayne, Ph.D.
Manager, Confocal and Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center, Columbia University
1130 St Nicholas Ave, 222A
New York, NY 10032

212-851-4613

[hidden email]
http://cancercenter.columbia.edu/confocal/

[elambeth]

Thank you that solved it (although for some reason the black was at 0, so I had to change the lower threshold to exclude my dark areas)

Emily Lambeth

Theresa Swayne wrote

Hi Emily,

If I understand the problem correctly, one way to solve it is:

-- (Image>Adjust>Threshold. Set upper threshold to 254. Lower  
threshold should be 0. Select "Over/Under" from the drop-down menu to  
see the excluded pixels in green.
-- Analyze > Set Measurements. Check "Mean" and "Limit to Threshold."
-- Analyze > Measure.

This will give you the mean of the pixels that are between 0 and 254,  
inclusive.

Hope this helps.
Theresa

On Aug 11, 2008, at 1:59 PM, elambeth wrote:

> I am working with red stained images of frog liver.  There are  
> pigment cells
> in the liver that are black, and should not count for the  
> quantification of
> the red stain.  I want to know the mean gray value of the red  
> pixels, and I
> know this is on the histogram, but I do not know how to exclude the  
> black
> pigment cells from the histogram (basically I want a histogram of  
> the red
> stain only).  Is there any way to exclude the black?
>

------------------------------------
Theresa C. Swayne, Ph.D.
Manager, Confocal and Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center, Columbia University
1130 St Nicholas Ave, 222A
New York, NY 10032

212-851-4613

[e-mail]
http://cancercenter.columbia.edu/confocal/

[Vergara, Leoncio A.]

In RGB images black will always be zero. The grayscale 0-255 corresponds to brightness in which white is 255. I think you need to be careful in interpreting your measurements, higher red intensities may correspond to lighter areas of the image (lack of stain) rather than more intense red staining.

You may have to convert your measurements to optical density so that darker red stain correspond to higher values.





Leoncio A. Vergara MD
Assistant Professor
Laboratory of Protein Misfolding Diseases (lab-PMD),
George and Cynthia Mitchell Center for Neurodegenerative Diseases Research.
Director of the Optical Imaging Lab. (OIL),
Dept. of Neuroscience and Cell Biology
University of Texas Medical Branch (UTMB)
301 University Blvd
Galveston, Texas 77555-0641
OIL phone: [phone]
Lab-PMD phone: [phone]
fax: [phone]


-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of elambeth
Sent: Monday, August 11, 2008 2:10 PM
To: [hidden email]
Subject: Re: Excluding black (255) from an image

Thank you that solved it (although for some reason the black was at 0, so I
had to change the lower threshold to exclude my dark areas)

Emily Lambeth

Theresa Swayne wrote:

>
> Hi Emily,
>
> If I understand the problem correctly, one way to solve it is:
>
> -- (Image>Adjust>Threshold. Set upper threshold to 254. Lower
> threshold should be 0. Select "Over/Under" from the drop-down menu to
> see the excluded pixels in green.
> -- Analyze > Set Measurements. Check "Mean" and "Limit to Threshold."
> -- Analyze > Measure.
>
> This will give you the mean of the pixels that are between 0 and 254,
> inclusive.
>
> Hope this helps.
> Theresa
>
> On Aug 11, 2008, at 1:59 PM, elambeth wrote:
>
>> I am working with red stained images of frog liver.  There are
>> pigment cells
>> in the liver that are black, and should not count for the
>> quantification of
>> the red stain.  I want to know the mean gray value of the red
>> pixels, and I
>> know this is on the histogram, but I do not know how to exclude the
>> black
>> pigment cells from the histogram (basically I want a histogram of
>> the red
>> stain only).  Is there any way to exclude the black?
>>
>
> ------------------------------------
> Theresa C. Swayne, Ph.D.
> Manager, Confocal and Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center, Columbia University
> 1130 St Nicholas Ave, 222A
> New York, NY 10032
>
> 212-851-4613
>
> [hidden email]
> http://cancercenter.columbia.edu/confocal/
>
>

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[elambeth]

Thank you again, I will calibrate to optical density standards.

Emily Lambeth

Vergara, Leoncio A. wrote

In RGB images black will always be zero. The grayscale 0-255 corresponds to brightness in which white is 255. I think you need to be careful in interpreting your measurements, higher red intensities may correspond to lighter areas of the image (lack of stain) rather than more intense red staining.

You may have to convert your measurements to optical density so that darker red stain correspond to higher values.





Leoncio A. Vergara MD
Assistant Professor
Laboratory of Protein Misfolding Diseases (lab-PMD),
George and Cynthia Mitchell Center for Neurodegenerative Diseases Research.
Director of the Optical Imaging Lab. (OIL),
Dept. of Neuroscience and Cell Biology
University of Texas Medical Branch (UTMB)
301 University Blvd
Galveston, Texas 77555-0641
OIL phone: [phone]
Lab-PMD phone: [phone]
fax: [phone]


-----Original Message-----
From: ImageJ Interest Group [mailto:[e-mail]] On Behalf Of elambeth
Sent: Monday, August 11, 2008 2:10 PM
To: [e-mail]
Subject: Re: Excluding black (255) from an image

Thank you that solved it (although for some reason the black was at 0, so I
had to change the lower threshold to exclude my dark areas)

Emily Lambeth

Theresa Swayne wrote:
>
> Hi Emily,
>
> If I understand the problem correctly, one way to solve it is:
>
> -- (Image>Adjust>Threshold. Set upper threshold to 254. Lower
> threshold should be 0. Select "Over/Under" from the drop-down menu to
> see the excluded pixels in green.
> -- Analyze > Set Measurements. Check "Mean" and "Limit to Threshold."
> -- Analyze > Measure.
>
> This will give you the mean of the pixels that are between 0 and 254,
> inclusive.
>
> Hope this helps.
> Theresa
>
> On Aug 11, 2008, at 1:59 PM, elambeth wrote:
>
>> I am working with red stained images of frog liver.  There are
>> pigment cells
>> in the liver that are black, and should not count for the
>> quantification of
>> the red stain.  I want to know the mean gray value of the red
>> pixels, and I
>> know this is on the histogram, but I do not know how to exclude the
>> black
>> pigment cells from the histogram (basically I want a histogram of
>> the red
>> stain only).  Is there any way to exclude the black?
>>
>
> ------------------------------------
> Theresa C. Swayne, Ph.D.
> Manager, Confocal and Specialized Microscopy Shared Resource
> Herbert Irving Comprehensive Cancer Center, Columbia University
> 1130 St Nicholas Ave, 222A
> New York, NY 10032
>
> 212-851-4613
>
> [e-mail]
> http://cancercenter.columbia.edu/confocal/
>
>

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