lamp temperature

[Kathleen McMillan]

Hello- I am looking for advice on whether I can correct microscope images for variations in lamp filament temperature when analyzing slides for stain intensity. In a first batch of slides, the background was white, the (unstained) tissue was tan and the stain blue.  Using the Landini thresholding plugin to select for blue hues followed by measuring the mean saturation worked well.  However, in a second batch of slides - which were intended to be combined with the first in the analysis - the slide background and the unstained tissue are a yellowish color and the stain hues are in the blue-magenta region.  That creates a problem in separating the tissue from the background, and also the mean saturation of the hues attributable to the stain are different from the first batch.  Is there a good way to correct for lighting variation in these two image sets, that would allow an accurate and consistent stain intensity to be quantified?  

Thanks for any advice!

[Richard Cole]

Switch to LED illumination.

Rich


Richard Cole
Research Scientist V
Director: Advanced Light Microscopy Core Unit
Wadsworth Center
 
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York

P.O. Box 509 Albany N.Y. 12201-0509
518-474-7048 Phone
518-474-4430 Fax

Email [hidden email]
Website www.wadsworth.org/cores/alm/index.htm



-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Kathleen McMillan
Sent: Thursday, June 02, 2011 2:04 PM
To: [hidden email]
Subject: lamp temperature

Hello- I am looking for advice on whether I can correct microscope images for variations in lamp filament temperature when analyzing slides for stain intensity. In a first batch of slides, the background was white, the (unstained) tissue was tan and the stain blue.  Using the Landini thresholding plugin to select for blue hues followed by measuring the mean saturation worked well.  However, in a second batch of slides - which were intended to be combined with the first in the analysis - the slide background and the unstained tissue are a yellowish color and the stain hues are in the blue-magenta region.  That creates a problem in separating the tissue from the background, and also the mean saturation of the hues attributable to the stain are different from the first batch.  Is there a good way to correct for lighting variation in these two image sets, that would allow an accurate and consistent stain intensity to be quantified?  

Thanks for any advice!



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[Joel Sheffield]

At the risk of blowing my own, old horn, plates 20-21 of a powerpoint
tutorial I presented at the Microscopy Meetings in '08 disscuss the issue of
white balance, and provide a manual way of doing it.
http://imagej.nih.gov/ij/docs/examples/IJ-M&M08.ppt  I am not aware of a
plugin or macro for that purpose, although it should be relatively easy to
implement.

Joel


On Thu, Jun 2, 2011 at 2:04 PM, Kathleen McMillan <
[hidden email]> wrote:

> Hello- I am looking for advice on whether I can correct microscope images
> for variations in lamp filament temperature when analyzing slides for stain
> intensity. In a first batch of slides, the background was white, the
> (unstained) tissue was tan and the stain blue.  Using the Landini
> thresholding plugin to select for blue hues followed by measuring the mean
> saturation worked well.  However, in a second batch of slides - which were
> intended to be combined with the first in the analysis - the slide
> background and the unstained tissue are a yellowish color and the stain hues
> are in the blue-magenta region.  That creates a problem in separating the
> tissue from the background, and also the mean saturation of the hues
> attributable to the stain are different from the first batch.  Is there a
> good way to correct for lighting variation in these two image sets, that
> would allow an accurate and consistent stain intensity to be quantified?
>
> Thanks for any advice!
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Nathaniel Ryckman]

Kathleen McMillan wrote

Hello- I am looking for advice on whether I can correct microscope images for variations in lamp filament temperature when analyzing slides for stain intensity. In a first batch of slides, the background was white, the (unstained) tissue was tan and the stain blue.  Using the Landini thresholding plugin to select for blue hues followed by measuring the mean saturation worked well.  However, in a second batch of slides - which were intended to be combined with the first in the analysis - the slide background and the unstained tissue are a yellowish color and the stain hues are in the blue-magenta region.  That creates a problem in separating the tissue from the background, and also the mean saturation of the hues attributable to the stain are different from the first batch.  Is there a good way to correct for lighting variation in these two image sets, that would allow an accurate and consistent stain intensity to be quantified?  

Thanks for any advice!

That sounds like a really tricky problem. I highly doubt that there is an easy solution.

The only things that I can think of is perhaps somehow normalizing colors across images by creating a custom plugin:

http://imagej.588099.n2.nabble.com/help-on-normalizing-background-across-pictures-td4893699.html

If you think it's because of uneven lighting, perhaps, you could split the color channels, perform intensity corrections (using rolling ball/flatfield), and then merge them back together:

http://www.macbiophotonics.ca/imagej/image_intensity_proce.htm

One last idea is that you could increase the threshold range to pick up colors from blue to blue-magenta (aka not be as selective about the regions that are automatically chosen).

[Gabriel Landini]

On Thursday 02 Jun 2011 19:04:27 you wrote:

> Hello- I am looking for advice on whether I can correct microscope images
> for variations in lamp filament temperature when analyzing slides for
> stain intensity. In a first batch of slides, the background was white, the
> (unstained) tissue was tan and the stain blue.  Using the Landini
> thresholding plugin to select for blue hues followed by measuring the mean
> saturation worked well.  However, in a second batch of slides - which were
> intended to be combined with the first in the analysis - the slide
> background and the unstained tissue are a yellowish color and the stain
> hues are in the blue-magenta region.  That creates a problem in separating
> the tissue from the background, and also the mean saturation of the hues
> attributable to the stain are different from the first batch.  Is there a
> good way to correct for lighting variation in these two image sets, that
> would allow an accurate and consistent stain intensity to be quantified?

It is possible to compensate for that and uneven illumination at the same
time. This is the traditional way of compensating illumination via
transmittance:
http://imagejdocu.tudor.lu/doku.php?id=howto:working:how_to_correct_background_illumination_in_brightfield_microscopy

The other problems with stains are:
The colour threshold plugin would work only if the stains do not colocalise.
For instance it is virtually impossible to be able to threshold H&E this way
because many pixels will record a combination of both dyes.

You might want to try stain separation using colour deconvolution, but be
aware that if the stains are not stoichiometric (most are not) then the
intensity of the stain will not be a quantitative measurement.

Cheers

Gabriel

[Kathleen McMillan]

Gabriel - thank you, this document is tremendously helpful for background
correction.
Kathleen

 Kathleen McMillan, PhD
President
gRadiant Research
1958 Main Street
Concord MA 01742




________________________________
From: Gabriel Landini <[hidden email]>
To: [hidden email]
Sent: Fri, June 3, 2011 4:00:54 AM
Subject: Re: lamp temperature

On Thursday 02 Jun 2011 19:04:27 you wrote:

> Hello- I am looking for advice on whether I can correct microscope images
> for variations in lamp filament temperature when analyzing slides for
> stain intensity. In a first batch of slides, the background was white, the
> (unstained) tissue was tan and the stain blue.  Using the Landini
> thresholding plugin to select for blue hues followed by measuring the mean
> saturation worked well.  However, in a second batch of slides - which were
> intended to be combined with the first in the analysis - the slide
> background and the unstained tissue are a yellowish color and the stain
> hues are in the blue-magenta region.  That creates a problem in separating
> the tissue from the background, and also the mean saturation of the hues
> attributable to the stain are different from the first batch.  Is there a
> good way to correct for lighting variation in these two image sets, that
> would allow an accurate and consistent stain intensity to be quantified?

It is possible to compensate for that and uneven illumination at the same
time. This is the traditional way of compensating illumination via
transmittance:
http://imagejdocu.tudor.lu/doku.php?id=howto:working:how_to_correct_background_illumination_in_brightfield_microscopy


The other problems with stains are:
The colour threshold plugin would work only if the stains do not colocalise.
For instance it is virtually impossible to be able to threshold H&E this way
because many pixels will record a combination of both dyes.

You might want to try stain separation using colour deconvolution, but be
aware that if the stains are not stoichiometric (most are not) then the
intensity of the stain will not be a quantitative measurement.

Cheers

Gabriel