help for background subtraction

[chen li]

Hi all,

I have a jpeg image of Western blot with high background and some ugly dots. I
wonder how good it can be if

the background  and the big dots(if possible) are removed using ImageJ. Since I
have no experience using the background subtraction plugin/function,
I wonder if someone can do me the favor. If you agree, I will send you the
image.


Thanks in advance,


Li

[John Oreopoulos]

Li, without knowing a bit more about the image quality and how it was acquired, it's hard to say, but I would advise against outright removal of sample artifacts (big dots). That might be considered a form of manipulation. I don't have much experience with westerns and I don't know what's considered acceptable post-processing, so I can't say for sure. The issue is much more clear-cut in biological microscopy. An article by Rossner in the Journal of Cell Biology clearly lays out some guidelines for both gel and microscopy images:

Rossner, M. and K.M. Yamada, What's in a picture? The temptation of image manipulation. Journal of Cell Biology, 2004. 166(1): p. 11-15.

A more recent publication by Cromey goes into a bit more detail:

Cromey, D., Avoiding twisted pixels: Ethical guidelines for the appropriate use and manipulation of scientific digital images. Science and Engineering Ethics, 2010.

Here are a few clippings from that article:

"Manipulations that are specific to one area of an Image and are not performed on other areas are questionable...
This includes image processing procedures that are analogous to the darkroom techniques of dodging and burning. Purists believe that selective enhancement should never be performed. However, there are rare occasions when it may be legitimate to enhance a specific area in an image. An example of a legitimate use of selective enhancement is a 16-bit greyscale image that has important features at both extremes of its dynamic range. To enable readers to see both features, a scientist might decide to selectively enhance the image. If portions of a published image are selectively enhanced, however, the author must clearly state this in the figure legend. Selective enhancement of an image that is not reported would most likely be viewed as research misconduct. Nearly every other type of selective image enhancement would probably be considered inappropriate. This includes the selective enhancement of specific bands or lanes in gel images. Some types of software filters for digital images can be selective in their application within an image (Russ 1998)."

"Cropping is usually considered an acceptable form of image manipulation (MacKenzie et al. 2006; Rossner and Yamada 2004). When cropping an image, a researcher should carefully examine his or her motivation. Is the crop intended to improve the ‘‘composition’’ of the image? Or is it being used to remove something that they do not like or understand in the image, or to hide something that disagrees with their lab’s preferred hypothesis? Legitimate reasons for cropping include centering an object of interest, trimming ‘‘empty’’ space around the edges of an image, and excluding a piece of debris. Questionable forms of cropping include removing information in a way that changes the context of what remains in the image after it has been cropped. An example of changing the context would be cropping out dead or dying cells to only display a healthy cell, or cropping out gel bands that might disagree with the hypothesis being proposed in the paper."

If possible, you may be better off trying to repeat the experiment so that these troublesome "dots" don't appear in a cleaner preparation.

As for general background signal removal, as long as you subtract the same value from all parts of the image of the gel, and apply the same procedure to all your gel images, this should be acceptable. Just be sure to write down all the image post-processing details you choose to follow. See this link for a good tutorial:

http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis

John Oreopoulos




On 2011-01-06, at 6:09 PM, chen li wrote:

> Hi all,
>
> I have a jpeg image of Western blot with high background and some ugly dots. I
> wonder how good it can be if
>
> the background  and the big dots(if possible) are removed using ImageJ. Since I
> have no experience using the background subtraction plugin/function,
> I wonder if someone can do me the favor. If you agree, I will send you the
> image.
>
>
> Thanks in advance,
>
>
> Li
>
>
>

[Johannes-P. Koch]

Li,

as far as I know, ugly dots in a western are very likely correlated with
a problem in blocking (milk not thoroughly dissolved or any other
reason). Sometimes other reasons might be true. If these dots occur,
they usually don't "destroy" the result, they are just ugly.

However, if you remove the dots it could be that you run into problems -
I agree with John's posting. You would have to state in the legend that
you removed some dots which is problematic of course.

Our lab policy is very clear-cut: Either you live with your dots or - in
case you want your perfect Nature publication - go back to the bench and
reload or redo the experiment (which can be troublesome of course).

Best,

Johannes

Am 07.01.2011 00:51, schrieb John Oreopoulos:

> Li, without knowing a bit more about the image quality and how it was acquired, it's hard to say, but I would advise against outright removal of sample artifacts (big dots). That might be considered a form of manipulation. I don't have much experience with westerns and I don't know what's considered acceptable post-processing, so I can't say for sure. The issue is much more clear-cut in biological microscopy. An article by Rossner in the Journal of Cell Biology clearly lays out some guidelines for both gel and microscopy images:
>
> Rossner, M. and K.M. Yamada, What's in a picture? The temptation of image manipulation. Journal of Cell Biology, 2004. 166(1): p. 11-15.
>
> A more recent publication by Cromey goes into a bit more detail:
>
> Cromey, D., Avoiding twisted pixels: Ethical guidelines for the appropriate use and manipulation of scientific digital images. Science and Engineering Ethics, 2010.
>
> Here are a few clippings from that article:
>
> "Manipulations that are specific to one area of an Image and are not performed on other areas are questionable...
> This includes image processing procedures that are analogous to the darkroom techniques of dodging and burning. Purists believe that selective enhancement should never be performed. However, there are rare occasions when it may be legitimate to enhance a specific area in an image. An example of a legitimate use of selective enhancement is a 16-bit greyscale image that has important features at both extremes of its dynamic range. To enable readers to see both features, a scientist might decide to selectively enhance the image. If portions of a published image are selectively enhanced, however, the author must clearly state this in the figure legend. Selective enhancement of an image that is not reported would most likely be viewed as research misconduct. Nearly every other type of selective image enhancement would probably be considered inappropriate. This includes the selective enhancement of specific bands or lanes in gel images. Some types of software filters for digital images can be selective in their application within an image (Russ 1998)."
>
> "Cropping is usually considered an acceptable form of image manipulation (MacKenzie et al. 2006; Rossner and Yamada 2004). When cropping an image, a researcher should carefully examine his or her motivation. Is the crop intended to improve the ‘‘composition’’ of the image? Or is it being used to remove something that they do not like or understand in the image, or to hide something that disagrees with their lab’s preferred hypothesis? Legitimate reasons for cropping include centering an object of interest, trimming ‘‘empty’’ space around the edges of an image, and excluding a piece of debris. Questionable forms of cropping include removing information in a way that changes the context of what remains in the image after it has been cropped. An example of changing the context would be cropping out dead or dying cells to only display a healthy cell, or cropping out gel bands that might disagree with the hypothesis being proposed in the paper."
>
> If possible, you may be better off trying to repeat the experiment so that these troublesome "dots" don't appear in a cleaner preparation.
>
> As for general background signal removal, as long as you subtract the same value from all parts of the image of the gel, and apply the same procedure to all your gel images, this should be acceptable. Just be sure to write down all the image post-processing details you choose to follow. See this link for a good tutorial:
>
> http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis
>
> John Oreopoulos
>
>
>
>
> On 2011-01-06, at 6:09 PM, chen li wrote:
>
>> Hi all,
>>
>> I have a jpeg image of Western blot with high background and some ugly dots. I
>> wonder how good it can be if
>>
>> the background  and the big dots(if possible) are removed using ImageJ. Since I
>> have no experience using the background subtraction plugin/function,
>> I wonder if someone can do me the favor. If you agree, I will send you the
>> image.
>>
>>
>> Thanks in advance,
>>
>>
>> Li
>>
>>
>>

--
Mag. Johannes-P. KOCH
Department of Biochemistry and Cell Biology
MFPL, University of Vienna
Dr. Bohrgasse 9/5
A-1030 Vienna
Austria

phone: [phone]
fax: [phone]

mail to: [hidden email]

[chen li]

Thanks but do you mind seeing the picture first?


Li



----- Original Message ----
From: Johannes-P. Koch <[hidden email]>
To: [hidden email]
Sent: Fri, January 7, 2011 2:06:29 AM
Subject: Re: help for background subtraction

Li,

as far as I know, ugly dots in a western are very likely correlated with
a problem in blocking (milk not thoroughly dissolved or any other
reason). Sometimes other reasons might be true. If these dots occur,
they usually don't "destroy" the result, they are just ugly.

However, if you remove the dots it could be that you run into problems -
I agree with John's posting. You would have to state in the legend that
you removed some dots which is problematic of course.

Our lab policy is very clear-cut: Either you live with your dots or - in
case you want your perfect Nature publication - go back to the bench and
reload or redo the experiment (which can be troublesome of course).

Best,

Johannes

Am 07.01.2011 00:51, schrieb John Oreopoulos:

> Li, without knowing a bit more about the image quality and how it was acquired,
>it's hard to say, but I would advise against outright removal of sample
>artifacts (big dots). That might be considered a form of manipulation. I don't
>have much experience with westerns and I don't know what's considered acceptable
>post-processing, so I can't say for sure. The issue is much more clear-cut in
>biological microscopy. An article by Rossner in the Journal of Cell Biology
>clearly lays out some guidelines for both gel and microscopy images:
>
> Rossner, M. and K.M. Yamada, What's in a picture? The temptation of image
>manipulation. Journal of Cell Biology, 2004. 166(1): p. 11-15.
>
> A more recent publication by Cromey goes into a bit more detail:
>
> Cromey, D., Avoiding twisted pixels: Ethical guidelines for the appropriate use
>and manipulation of scientific digital images. Science and Engineering Ethics,
>2010.
>
> Here are a few clippings from that article:
>
> "Manipulations that are specific to one area of an Image and are not performed
>on other areas are questionable...
> This includes image processing procedures that are analogous to the darkroom
>techniques of dodging and burning. Purists believe that selective enhancement
>should never be performed. However, there are rare occasions when it may be
>legitimate to enhance a specific area in an image. An example of a legitimate
>use of selective enhancement is a 16-bit greyscale image that has important
>features at both extremes of its dynamic range. To enable readers to see both
>features, a scientist might decide to selectively enhance the image. If portions
>of a published image are selectively enhanced, however, the author must clearly
>state this in the figure legend. Selective enhancement of an image that is not
>reported would most likely be viewed as research misconduct. Nearly every other
>type of selective image enhancement would probably be considered inappropriate.
>This includes the selective enhancement of specific bands or lanes in gel
>images. Some types of software filters for digital images can be selective in
>their application within an image (Russ 1998)."
>
> "Cropping is usually considered an acceptable form of image manipulation
>(MacKenzie et al. 2006; Rossner and Yamada 2004). When cropping an image, a
>researcher should carefully examine his or her motivation. Is the crop intended
>to improve the ‘‘composition’’ of the image? Or is it being used to remove
>something that they do not like or understand in the image, or to hide something
>that disagrees with their lab’s preferred hypothesis? Legitimate reasons for
>cropping include centering an object of interest, trimming ‘‘empty’’ space
>around the edges of an image, and excluding a piece of debris. Questionable
>forms of cropping include removing information in a way that changes the context
>of what remains in the image after it has been cropped. An example of changing
>the context would be cropping out dead or dying cells to only display a healthy
>cell, or cropping out gel bands that might disagree with the hypothesis being
>proposed in the paper."
>
> If possible, you may be better off trying to repeat the experiment so that
>these troublesome "dots" don't appear in a cleaner preparation.
>
> As for general background signal removal, as long as you subtract the same
>value from all parts of the image of the gel, and apply the same procedure to
>all your gel images, this should be acceptable. Just be sure to write down all
>the image post-processing details you choose to follow. See this link for a good
>tutorial:
>
>http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis
>s
>
> John Oreopoulos
>
>
>
>
> On 2011-01-06, at 6:09 PM, chen li wrote:
>
>> Hi all,
>>
>> I have a jpeg image of Western blot with high background and some ugly dots.

I

>> wonder how good it can be if
>>
>> the background  and the big dots(if possible) are removed using ImageJ. Since
>I
>> have no experience using the background subtraction plugin/function,
>> I wonder if someone can do me the favor. If you agree, I will send you the
>> image.
>>
>>
>> Thanks in advance,
>>
>>
>> Li
>>
>>
>>

--
Mag. Johannes-P. KOCH
Department of Biochemistry and Cell Biology
MFPL, University of Vienna
Dr. Bohrgasse 9/5
A-1030 Vienna
Austria

phone: [phone]
fax: [phone]

mail to: [hidden email]

[albert]

Hi John,
I want to find the intensity of diffraction spots in a stack of images of diffraction patterns. Could you help me how the background correction is done in this case? i could not find any information regarding this problem in the imagj's manual.

thanks
albert