ImageJ - Re: Cell counting with imageJ

ImageJ

Re: Cell counting with imageJ

Posted by Lukas Hoffmann on
URL: http://imagej.273.s1.nabble.com/Cell-counting-with-imageJ-tp3690375p3690384.html

To All,

I am currently writing just such a program for
autocounting fluorescently stained cells. I will be happy to give you
this program for free when I complete it, if you request.
But, I do not know when it will be complete. I am an undergrad working
20 hrs a week who is going to take time off for christmas break. The project is in its early stages therefore I cannot predict if
I will have success. I am no expert on this subject. But there is no
problem for you if I fail, of course.

My
basic idea is, you count a few images by hand and tell the program what
the count "should" be. Then the program changes the way it does the
thresholding & image processing in many different ways, and
compares all of its test counts with the correct count. When the
program finds one type of image processing that has a low error rate
compared to the manual counting, it uses that type of processing for
the rest of your images.

For this to work, all of your images
must be similarly obtained: same type of cells, same lighting
conditions, etc. If you have significantly different images, then the
tests have to be re run.

If you are interested in collaborating,
please send me some of your images that you have manually counted. I
can use them to refine my program so that it works on my lab's images
and your lab's images.

Lukas Hoffmann
Carnegie Mellon University
Center for the Neural Basis of Cognition
Urban Lab

> Date: Fri, 13 Nov 2009 10:26:21 -0500
> From: [hidden email]
> Subject: Re: Cell counting with imageJ
> To: [hidden email]
>
> Esteban: I think ITCN uses a process somewhat more complex than watershed.
> It does not threshold the image, so it works quite well for counting nuclei
> that are very close together, and even overlapping slightly. As I
> understand, it uses the colvolution and find maxima functions instead. It
> requires a little trial and error in setting up the input parameters
> initially, but then the process moves pretty quickly, and it produces better
> results than I ever managed using thresholding and watershed processing. It
> also marks the image so that you can do some quality control.
>
> Antonis: It is a little picky--make sure the image is open before you try to
> launch ITCN. Also, optimize the input parameters with a smaller ROI--if your
> images are large, it takes a lot of processing power.
>
> Jason
>
>
> On Fri, Nov 13, 2009 at 9:46 AM, G. Esteban Fernandez <
> [hidden email]> wrote:
>
> > You can also use the the built-in functions under Analyze > Analyze
> > Particles to count automatically after thresholding the images. If
> > thresholding doesn't do a good job of including all the nuclei and
> > excluding background then it can get tricky. Good thresholding is the
> > key to good results with automatic counting. Also, if there are
> > nuclei touching each other you can separate them with Process > Binary
> > > Watershed. Maybe the ITCN plugin does the separation automatically?
> >
> > -Esteban
> >
> >
> > On Fri, Nov 13, 2009 at 8:36 AM, Jason Bailey <[hidden email]>
> > wrote:
> > > Antonis,
> > >
> > > I have had much luck with the Image-Based Tool for Counting Nuclei (ITCN)
> > > with mammalian cells. I imagine it could work well with yours as well. I
> > > typically do two counts--first count all nuclei labeled with Hoechst
> > 33342,
> > > then count the nuclei labeled with a different dye (either ethidium or
> > FITC,
> > > usually). Although this might require some changes to how you label your
> > > cells.
> > >
> > > If you must resort to manual counting, the "Cell Counter" plugin marks
> > > cells/nuclei as you click and lets you track multiple cell types as you
> > go.
> > >
> > > Both are available at the ImageJ Plugins page:
> > > http://rsbweb.nih.gov/ij/plugins/index.html
> > >
> > > Good luck!
> > >
> > > Jason
> > >
> > > On Fri, Nov 13, 2009 at 9:25 AM, Antonis Giakountis <[hidden email]>
> > wrote:
> > >
> > >> Hi Esteban,
> > >>
> > >> I really would prefer not to do it by hand. A typical experiment of mine
> > >> involves several timepoints (10-16) and at least five reps per line.
> > Each
> > >> one of those has hundreds of cells and I was hoping for some stitistics
> > out
> > >> of this. To make a long story sort I expect thousants of cells and I
> > would
> > >> like to get an estimation of the percentage of them that has
> > fluorescence
> > >> out of the total.
> > >>
> > >> In the worst case I could deal with user define-clinking as long as I
> > could
> > >> mark the cells that are counted. As for the images I am aiming for
> > >> triple-stain nuclear immunofluorescence, but since this is done with the
> > >> confocal I can easily scan each channel separately, including the
> > >> transmitted-light one (BF).
> > >>
> > >> Antonis
> > >> ----- Original Message ----- From: "G. Esteban Fernandez" <
> > >> [hidden email]>
> > >>
> > >> To: <[hidden email]>
> > >> Sent: Friday, November 13, 2009 2:10 PM
> > >>
> > >> Subject: Re: Cell counting with imageJ
> > >>
> > >>
> > >> How many images/cells do you need to count? Is this something that
> > >>> you can do by hand in a matter of a couple of days or do you want an
> > >>> automated procedure? Often it takes a few days to get an automated
> > >>> procedure to work correctly, if it ever works at all, during which
> > >>> time you could've done the counting by hand. There are probably
> > >>> ImageJ plugins that keep track of counts as you click on cells in the
> > >>> images.
> > >>>
> > >>> Do you have images that match the fluorescence images to get total
> > >>> cell numbers? For example transmitted light (brightfield/phase/DIC)
> > >>> images?
> > >>>
> > >>> -Esteban
> > >>>
> > >>>
> > >>> On Fri, Nov 13, 2009 at 7:43 AM, Antonis Giakountis <[hidden email]>
> > >>> wrote:
> > >>>
> > >>>> Dear all,
> > >>>>
> > >>>> I am a plant Biologist and I would like to use ImageJ in order to
> > count
> > >>>> cell numbers (diameter 10-20 microns on average) and to estimate
> > percentage
> > >>>> of cells that have fluorescence.
> > >>>>
> > >>>> Is it possible to to that with ImageJ and if yes do I have to install
> > a
> > >>>> certain plugin? If not can you recomment another software?
> > >>>>
> > >>>> Thank you in advance for your time,
> > >>>>
> > >>>> Antonis
> > >>>>
> > >>>> --------------------------------------------
> > >>>> Antonis Giakountis, PhD
> > >>>> Prof. David Baulcombe group
> > >>>> Department of Plant Sciences
> > >>>> University of Cambridge
> > >>>> Downing Street
> > >>>> Cambridge CB2 3EA
> > >>>> UK
> > >>>>
> > >>>>
> > >>>
> > >>>
> > >>> --
> > >>> G. Esteban Fernandez, Ph.D.
> > >>>
> > >>> Associate Director
> > >>> Molecular Cytology Core Facility
> > >>> University of Missouri
> > >>> 120 Bond Life Sciences Center
> > >>> Columbia, MO 65211
> > >>>
> > >>> http://www.biotech.missouri.edu/mcc
> > >>>
> > >>> (573)882-4895
> > >>> (573)884-9676 fax
> > >>>
> > >>>
> > >
> >
> >
> >
> > --
> > G. Esteban Fernandez, Ph.D.
> >
> > Associate Director
> > Molecular Cytology Core Facility
> > University of Missouri
> > 120 Bond Life Sciences Center
> > Columbia, MO 65211
> >
> > http://www.biotech.missouri.edu/mcc
> >
> > (573)882-4895
> > (573)884-9676 fax
> >

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