Pls help me with segmentation issue to count cell colonies in 3D agars.

[Alan Thames]

Dear ImageJ users,
                             I have an issue with counting my cell colonies
in 3D agars. I have attached 8 bit images, in which you can see control image
has huge and multiple colonies, while the other image  treated with a drug,
 50 uM concentation has  multiple tiny
colonies. When i try to count the colonies number with imageJ, i found
 the count
" number" is even  more increased  in treated images, which i do not want
to see,obviously, though the size of the colonies are counted to be
decreased with increasing drug concentration ( pls see below results;
settings - Image>Adjust>Threshold>Auto, Analyze>Analyze Particles, Size: 0
to infinity, circularity 0-1 and Exclude on Edges)  . As one suggested, the
colonies are all in different planes (ie the agar is too tick to b enabling
focusing
in all colonies at once). That will prevent segmenting them right as there are
too many halo (out of focus) artifacts. Can any one help me with this
please?


                        Count               total Area         Average Size
       % area
Control:             4164 398785.000000 95.769693 12.482002
50 uM :             9691   194228.000000 20.042101 6.079352


I appreciate it.

Regards,
Alan

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[Peter Haub]

Hi Alan,

you can try to use a minimum filter (Process > Filters > Minimum ..).
Maybe this can help to separate (and count) the colonies.

In any case you should do a flat field correction.

And maybe there is a chance to improve the imaging. What's your optical
system?

Regards,
Peter

On 30.09.2013 18:07, Alan Thames wrote:

> Dear ImageJ users,
>                               I have an issue with counting my cell colonies
> in 3D agars. I have attached 8 bit images, in which you can see control image
> has huge and multiple colonies, while the other image  treated with a drug,
>   50 uM concentation has  multiple tiny
> colonies. When i try to count the colonies number with imageJ, i found
>   the count
> " number" is even  more increased  in treated images, which i do not want
> to see,obviously, though the size of the colonies are counted to be
> decreased with increasing drug concentration ( pls see below results;
> settings - Image>Adjust>Threshold>Auto, Analyze>Analyze Particles, Size: 0
> to infinity, circularity 0-1 and Exclude on Edges)  . As one suggested, the
> colonies are all in different planes (ie the agar is too tick to b enabling
> focusing
> in all colonies at once). That will prevent segmenting them right as there are
> too many halo (out of focus) artifacts. Can any one help me with this
> please?
>
>
>                          Count               total Area         Average Size
>         % area
> Control:             4164 398785.000000 95.769693 12.482002
> 50 uM :             9691   194228.000000 20.042101 6.079352
>
>
> I appreciate it.
>
> Regards,
> Alan
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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[Alan Thames]

Hi Peter,
               I used Nikon Eclipse TS100 microscope with Inifinity 2
capturing tool from Micron-Optics. I took these pictures months ago and
saved as jpeg formats.  Where can i find how to do a flat field correction?

Thank you.

Alan


On Mon, Sep 30, 2013 at 2:12 PM, Peter Haub <[hidden email]> wrote:

> Hi Alan,
>
> you can try to use a minimum filter (Process > Filters > Minimum ..).
> Maybe this can help to separate (and count) the colonies.
>
> In any case you should do a flat field correction.
>
> And maybe there is a chance to improve the imaging. What's your optical
> system?
>
> Regards,
> Peter
>
>
> On 30.09.2013 18:07, Alan Thames wrote:
>
>> Dear ImageJ users,
>>                               I have an issue with counting my cell
>> colonies
>> in 3D agars. I have attached 8 bit images, in which you can see control
>> image
>> has huge and multiple colonies, while the other image  treated with a
>> drug,
>>   50 uM concentation has  multiple tiny
>> colonies. When i try to count the colonies number with imageJ, i found
>>   the count
>> " number" is even  more increased  in treated images, which i do not want
>> to see,obviously, though the size of the colonies are counted to be
>> decreased with increasing drug concentration ( pls see below results;
>> settings - Image>Adjust>Threshold>Auto, Analyze>Analyze Particles, Size: 0
>> to infinity, circularity 0-1 and Exclude on Edges)  . As one suggested,
>> the
>> colonies are all in different planes (ie the agar is too tick to b
>> enabling
>> focusing
>> in all colonies at once). That will prevent segmenting them right as
>> there are
>> too many halo (out of focus) artifacts. Can any one help me with this
>> please?
>>
>>
>>                          Count               total Area         Average
>> Size
>>         % area
>> Control:             4164 398785.000000 95.769693 12.482002
>> 50 uM :             9691   194228.000000 20.042101 6.079352
>>
>>
>> I appreciate it.
>>
>> Regards,
>> Alan
>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>

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[Peter Haub]

Hi Alan,

you can find various infos about shading correction (or flat field
correction) in the net. Search for"ImageJ shading correction".
Examples are:
http://rsbweb.nih.gov/ij/plugins/shading-corrector.html
http://imagejdocu.tudor.lu/doku.php?id=howto:working:how_to_correct_background_illumination_in_brightfield_microscopy

The background in your images are so inhomogeneous that thresholding
will lead to problems.
The problem will be that you do not have a background image since the
images are captured month ago - as you wrote.

The images look like phase contrast. I would try to eliminate the ring
artefact around the objects. But this is an academic discussion if there
is no chance to recapture the samples.

Just one more hint: Saving scientific images in jpg-format is not
optimal. You will lose information and introduce jpg-artefact which are
clearly visible in your images and which are sub-optimal for every
analysis process.

Regards,
Peter

On 02.10.2013 21:44, Alan Thames wrote:

> Hi Peter,
>                 I used Nikon Eclipse TS100 microscope with Inifinity 2
> capturing tool from Micron-Optics. I took these pictures months ago and
> saved as jpeg formats.  Where can i find how to do a flat field correction?
>
> Thank you.
>
> Alan
>
>
> On Mon, Sep 30, 2013 at 2:12 PM, Peter Haub <[hidden email]> wrote:
>
>> Hi Alan,
>>
>> you can try to use a minimum filter (Process > Filters > Minimum ..).
>> Maybe this can help to separate (and count) the colonies.
>>
>> In any case you should do a flat field correction.
>>
>> And maybe there is a chance to improve the imaging. What's your optical
>> system?
>>
>> Regards,
>> Peter
>>
>>
>> On 30.09.2013 18:07, Alan Thames wrote:
>>
>>> Dear ImageJ users,
>>>                                I have an issue with counting my cell
>>> colonies
>>> in 3D agars. I have attached 8 bit images, in which you can see control
>>> image
>>> has huge and multiple colonies, while the other image  treated with a
>>> drug,
>>>    50 uM concentation has  multiple tiny
>>> colonies. When i try to count the colonies number with imageJ, i found
>>>    the count
>>> " number" is even  more increased  in treated images, which i do not want
>>> to see,obviously, though the size of the colonies are counted to be
>>> decreased with increasing drug concentration ( pls see below results;
>>> settings - Image>Adjust>Threshold>Auto, Analyze>Analyze Particles, Size: 0
>>> to infinity, circularity 0-1 and Exclude on Edges)  . As one suggested,
>>> the
>>> colonies are all in different planes (ie the agar is too tick to b
>>> enabling
>>> focusing
>>> in all colonies at once). That will prevent segmenting them right as
>>> there are
>>> too many halo (out of focus) artifacts. Can any one help me with this
>>> please?
>>>
>>>
>>>                           Count               total Area         Average
>>> Size
>>>          % area
>>> Control:             4164 398785.000000 95.769693 12.482002
>>> 50 uM :             9691   194228.000000 20.042101 6.079352
>>>
>>>
>>> I appreciate it.
>>>
>>> Regards,
>>> Alan
>>>
>>> --
>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>>
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> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[dscho]

Hi Peter,

On Fri, 4 Oct 2013, Peter Haub wrote:

> Just one more hint: Saving scientific images in jpg-format is not
> optimal. You will lose information and introduce jpg-artefact which are
> clearly visible in your images and which are sub-optimal for every
> analysis process.

Often, those artifacts are not so clearly visible (JPEG format was
designed to fool our eyes, after all...) unless after processing -- and
then often only to the trained eye.

But artifacts they are, therefore JPEG is a poor format to store
scientific images in. It is appropriate only in a very narrow set of
bandwidth-constrained visualization tasks, and then only for transmitting,
never for storing nor analyzing.

Ciao,
Johannes

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[Shameem Sampath]

Hi Alan,
I've come across a paper where a similar situation with the formation of Small Colony Variants was seen and analysed with ImageJ


http://jmm.sgmjournals.org/content/59/5/521.long


regards,
Shameem Sampath MD
The Bluespot Knee Clinic
On 30 Sep 2013, at 17:07, Alan Thames wrote:

> Dear ImageJ users,
>                             I have an issue with counting my cell colonies
> in 3D agars. I have attached 8 bit images, in which you can see control image
> has huge and multiple colonies, while the other image  treated with a drug,
> 50 uM concentation has  multiple tiny
> colonies. When i try to count the colonies number with imageJ, i found
> the count
> " number" is even  more increased  in treated images, which i do not want
> to see,obviously, though the size of the colonies are counted to be
> decreased with increasing drug concentration ( pls see below results;
> settings - Image>Adjust>Threshold>Auto, Analyze>Analyze Particles, Size: 0
> to infinity, circularity 0-1 and Exclude on Edges)  . As one suggested, the
> colonies are all in different planes (ie the agar is too tick to b enabling
> focusing
> in all colonies at once). That will prevent segmenting them right as there are
> too many halo (out of focus) artifacts. Can any one help me with this
> please?
>
>
>                        Count               total Area         Average Size
>       % area
> Control:             4164 398785.000000 95.769693 12.482002
> 50 uM :             9691   194228.000000 20.042101 6.079352
>
>
> I appreciate it.
>
> Regards,
> Alan
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> <control.jpg><50 micromolar.jpg>

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[Peter Haub]

Hi Johannes,

Thanks for your comments and correction about artifacts in JPEG images.
You are right. 'Clearly' was not that clear.
I wish there would be a link to some background information on this
topic. And more a kind of "Do's and Dont's in Scientific Imaging" where
beginners could be guided to and where they can find the most relevant
and important info in short.

Do you have anymore idea beside the very nice books of Burger&Burge (
http://imagingbook.com ) and also Peter Bankhead (
http://blogs.qub.ac.uk/ccbg/files/2013/06/Analyzing_fluorescence_microscopy_images.pdf 
), where the aspects of jpeg compression is described?

Regards,
Peter

On 04.10.2013 14:50, Johannes Schindelin wrote:

> Hi Peter,
>
> On Fri, 4 Oct 2013, Peter Haub wrote:
>
>> Just one more hint: Saving scientific images in jpg-format is not
>> optimal. You will lose information and introduce jpg-artefact which are
>> clearly visible in your images and which are sub-optimal for every
>> analysis process.
> Often, those artifacts are not so clearly visible (JPEG format was
> designed to fool our eyes, after all...) unless after processing -- and
> then often only to the trained eye.
>
> But artifacts they are, therefore JPEG is a poor format to store
> scientific images in. It is appropriate only in a very narrow set of
> bandwidth-constrained visualization tasks, and then only for transmitting,
> never for storing nor analyzing.
>
> Ciao,
> Johannes
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[dscho]

Hi Peter,

On Sun, 6 Oct 2013, Peter Haub wrote:

> I wish there would be a link to some background information on this
> topic. And more a kind of "Do's and Dont's in Scientific Imaging" where
> beginners could be guided to and where they can find the most relevant
> and important info in short.
>
> Do you have anymore idea beside the very nice books of Burger&Burge (
> http://imagingbook.com ) and also Peter Bankhead (
> http://blogs.qub.ac.uk/ccbg/files/2013/06/Analyzing_fluorescence_microscopy_images.pdf 
> ), where the aspects of jpeg compression is described?

I also like to point to
http://www.uab.edu/researchintegrityandimages/guidelines/list.html and
http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf (and I also like to
point out that scientific images are not, in fact, photographs:
http://www.4p8.com/eric.brasseur/gamma.html)

Ciao,
Johannes

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[ctrueden]

Hi Peter,

> I wish there would be a link to some background information on this
> topic. And more a kind of "Do's and Dont's in Scientific Imaging"
> where beginners could be guided to and where they can find the most
> relevant and important info in short.

There is the start of such a page on the Fiji wiki:
    http://fiji.sc/IP_Principles

Feel free to edit and flesh out to your heart's content!

Regards,
Curtis


On Mon, Oct 7, 2013 at 4:40 AM, Johannes Schindelin <
[hidden email]> wrote:

> Hi Peter,
>
> On Sun, 6 Oct 2013, Peter Haub wrote:
>
> > I wish there would be a link to some background information on this
> > topic. And more a kind of "Do's and Dont's in Scientific Imaging" where
> > beginners could be guided to and where they can find the most relevant
> > and important info in short.
> >
> > Do you have anymore idea beside the very nice books of Burger&Burge (
> > http://imagingbook.com ) and also Peter Bankhead (
> >
> http://blogs.qub.ac.uk/ccbg/files/2013/06/Analyzing_fluorescence_microscopy_images.pdf
> > ), where the aspects of jpeg compression is described?
>
> I also like to point to
> http://www.uab.edu/researchintegrityandimages/guidelines/list.html and
> http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf (and I also like to
> point out that scientific images are not, in fact, photographs:
> http://www.4p8.com/eric.brasseur/gamma.html)
>
> Ciao,
> Johannes
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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[Alan Thames]

thank you all.


On Mon, Oct 7, 2013 at 11:47 AM, Curtis Rueden <[hidden email]> wrote:

> Hi Peter,
>
> > I wish there would be a link to some background information on this
> > topic. And more a kind of "Do's and Dont's in Scientific Imaging"
> > where beginners could be guided to and where they can find the most
> > relevant and important info in short.
>
> There is the start of such a page on the Fiji wiki:
>     http://fiji.sc/IP_Principles
>
> Feel free to edit and flesh out to your heart's content!
>
> Regards,
> Curtis
>
>
> On Mon, Oct 7, 2013 at 4:40 AM, Johannes Schindelin <
> [hidden email]> wrote:
>
> > Hi Peter,
> >
> > On Sun, 6 Oct 2013, Peter Haub wrote:
> >
> > > I wish there would be a link to some background information on this
> > > topic. And more a kind of "Do's and Dont's in Scientific Imaging" where
> > > beginners could be guided to and where they can find the most relevant
> > > and important info in short.
> > >
> > > Do you have anymore idea beside the very nice books of Burger&Burge (
> > > http://imagingbook.com ) and also Peter Bankhead (
> > >
> >
> http://blogs.qub.ac.uk/ccbg/files/2013/06/Analyzing_fluorescence_microscopy_images.pdf
> > > ), where the aspects of jpeg compression is described?
> >
> > I also like to point to
> > http://www.uab.edu/researchintegrityandimages/guidelines/list.html and
> > http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf (and I also like to
> > point out that scientific images are not, in fact, photographs:
> > http://www.4p8.com/eric.brasseur/gamma.html)
> >
> > Ciao,
> > Johannes
> >
> > --
> > ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> >
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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