How to make DNA ploidy analysis on Fleugen stained cells?

hello every one i am freshly interested one in image analysis and its techniques and i have some questions

how can i preform histological analysis such as DNA ploidy analysis,nuclear morphology and chromosome shape using cell profiler?

please i need this help in due course ? thanks to all in advance

Mahmoud
:stuck_out_tongue: :stuck_out_tongue: :stuck_out_tongue: :stuck_out_tongue: :stuck_out_tongue:

One point of clarification: Are you indeed using histological images or fluorescent images?

If the latter, then yes, CellProfiler can certainly extract measures of morphology and intensity from such images. You can take a look at this paper for a more sophisticated example.
-Mark

firstly ,i would like to thank you Mark on fast reply , secondly i am interested in histological images such as staining by Haematoxylin ,Toluidine blue ,Orcein stain ,Periodic acid-Schiff stain (PAS} and other stains types

isn’t there any way to use cell profiler in analyzing such images and why?
thanks in advance …

:unamused: :unamused: :unamused: :question: :question: :question: :question: :question: :question: :question: :question: :question: :question: :exclamation: :exclamation: :exclamation: :exclamation:

i am interested in histological images such as staining by Haematoxylin ,Toluidine blue ,Orcein stain ,Periodic acid-Schiff stain (PAS} and other stains types

isn’t there any way to use cell profiler in analyzing such images and why?
thanks in advance …
:bulb: :bulb: :bulb: :bulb:

Yes, you can still extract information from histological images, although the procedure tends to be more difficult than for fluorescent images. See this thread for an example using CellProfiler 1.0.

Regards,
-Mark

to be more precise about what i mean i have uploaded some pictures prepared by fleugen squash technique, please give me detailed step instructions helping me to determine DNA content in stained nuclei in these pictures"if possible as PDF document".

I have uploaded them on these links:
ifile.it/srmidu2/image%20analysis.JPG
ifile.it/qk9oerv/image%20analysis%20%281%29.JPG
ifile.it/cnusr4v/image%20analysis%20%282%29.JPG

another question :what are other possible info i can extract form such images?

thanks 8) 8) 8) 8) 8)

Hi,

I’m attaching a pipeline which can act as a starting point (it is capable with version r10211 which we just released). I’ve based the procedure from the paper at this link; if you google ‘Feulgen image analysis’, you can can find many others. It does the following:

  • Load the images and extracts the green channel

  • Inverts the green channel, identifies the DNA material, excluding those on the image border. Then it measures the intensity from the green channel for each object

  • Re-detects the DNA material, this time finding all cells, then masks the green channel with these objects and measures intensity in order to find the background intensity.

  • Calculates an approximate IOD from the integrated intensity of DNA pixels and the mean intensity from the background pixels. Please note that this is not
    the same as the formula in the above paper, since the log of a sum is not the same as the sum of logs. For a strictly correct formulation, you will probably need more ImageMath modules to do the transformations plus some knowledge of logarithmic operations.

  • Export the final results.

As far as other measurements go, you are free to use any of the other measurement modules on the detected DNA objects; I’ve added morphological and texture measures into the pipeline as well.

Regards,
-Mark
2010_07_19.cp (9.34 KB)

firstly i would like to express my sincere thanks for your help

  • secondly ,i am sorry i have little information in computational biology , can you please tell me what is the need for image math?
  • thirdly , i have used the pipeline but cell profiler analyst showed me an error message i think it may be from SQL,
    kindly look for the attached picture what is solve(ifile.it/skn3a8y/error.GIF)
  • why there is 2 software one for analysis and other for data?
  • is there any online courses for cell profiler etc… for international users?

thanks very much

In order to perform a mathematical calculation in image processing, you need the ability to perform mathematical operations on your images. So for example, if you wanted to take the logarithm of an image or the ratio of a couple of images, ImageMath allows you to do so.

[quote=“mahmoud”]thirdly , i have used the pipeline but cell profiler analyst showed me an error message i think it may be from SQL,
kindly look for the attached picture what is solve(ifile.it/skn3a8y/error.GIF)[/quote]

The error is generated because the properties file does not some of the fields correctly set, in this case, the database type; I wouldn’t be able to tell you more details without seeing the pipeline that generated the file. Even so, while we try to intelligently fill in as much of the settings in the properties file as we can, there are additional settings that would need to be filled in or checked manually. Your best course of action would be to take a look at the CPA manual, available from this page. However, based on the description of your project, you might be better served by using ExportToSpreadsheet in your pipeline and analyzing the output data in Excel or a similar package.

While we plan on integrating CellProfiler and CellProfiler Analyst in the future, the simple answer is that they do very different things; see question #2 in our FAQ.

As it turns out, there is not. However, we suggest making use of the example and tutorial webpages to help get started.

Regards,
-Mark

the pipeline i used is the one i downloaded from this site , is image math is included in the pipeline ?

plentiful thanks Mark
:unamused:

Hi,

ImageMath is included in the pipeline, for the purposes of inverting the intensity values. But it is capable of other arithmetic operations as well, as I mentioned before. You can add ImageMath (or any other module) by clicking the ‘+’ button below the module list in the main window, and you can experiment with changing the settings. Please feel free to modify the pipeline by adding modules and making adjustments as needed.

Regards,
-Mark

[size=200]thanks[/size]
8)
another question
how to identify G1, S and G2 phases using cell profiler, with same conditions

I’m not familiar with how the Feulgen stain is differentially stained in these stages, but I resume that as the DNA content doubles, the stain becomes darker. So after detecting the nuclei, you can use a MeasureObjectIntensity module to measure the intensity of the staining for each nuclei; several different intensity measurements are output, such as mean, median, integrated intensity etc. Then perhaps you can use ClassifyObjects to classify the nuclei into bins that you define on the basis of thresholds that you set.
-Mark