How to deal with the data of fly embryo staining?

Sample image and/or macro code

late stage and unfertilized eggs.tif (5.6 MB)

Background

This is a DAPI-staining picture about embryo of flies,.

Analysis goals

  • I would like to classify embryo in these images. As you know there are so many similiar pictures are going to deal with.

Challenges

  • It’s difficult to find the plugin to deal with this problem.
  • Some plugins.
  • Nope.
  • many but them didn’t work.

Hi,

it will help if you explain in more detail what you want to get out of the image. For example your example image - what is the desired result?

Blind guessing, but could Ilastik Object Classification be interesting for your data?

Hi,
Thank you for your advices. I just want to classify different embryso according their traits in about one hundred DAPI-staining images. And as you can see any image have 7 to10 embryos. Should I extrcat every embryo from the images first? And what’s the next step to classify different embryos. Thanks again for your reply.

Hi,

do you want to segment them, or just count them? How do you define the classes, by which properties?

If you need a segmentation you can either blur+threshold, then watershedding, remove wrong detections, then classify based on region properties. Or machine learning like with Ilastik object classification.
If you just want to count you might be similarly fast by manually counting the different classes with Imagej multipoint tool or Cell Counter.
it really depends on what you want to achieve exactly …

Other note: Your image is RGB and seems uncalibrated, is it maybe not the original image?

Hi,
Thank you for your reply. I guess segement every embryo from these images is the first step, and then I can classify every embryo into different stages of development. Also there are big differences of embryos which in different stages of development and it’s easy to tell in eyes.

Hi, noreenw,

Thank you so much for your advices. You have metioned that this picture is uncalibrated. Do you know how to calibrate this image? By the way, this image is filmed by CCD under a microscope.

Yours Sincerely,
NaN

Hi @NaN,

when you check under Image > Properties, you see that the unit is in pixels:

To answer biological questions you most likely what the unit to be um (or mm). This calibration is often stored in the meta-data. So in case you didn’t share the raw image it was maybe there originally and got lost in the share. You can also try Bio-Formats Importer for opening your image to try to recover the scale (when I try this I get 2.2587um/px - but no idea if this is correct).
If the image was never calibrated you’ll have to figure out the scale manually with e.g. a calibration slide for the microscope (glass slide that contains a scale). Most likely someone at your institute/university has done this before and can help.

Hi, noreenw,

Thank you for your advices. There are some problem when I want to open the images using Bio-Formats Importer:

ImageJ 2.1.0/1.53c; Java 1.8.0_172 [64-bit]; Windows 7 6.1; 48MB of 6041MB (<1%)

java.lang.NoClassDefFoundError: org/joda/time/ReadableInstant
at loci.formats.in.SISReader.initStandardMetadata(SISReader.java:170)
at loci.formats.in.BaseTiffReader.initMetadata(BaseTiffReader.java:97)
at loci.formats.in.BaseTiffReader.initFile(BaseTiffReader.java:586)
at loci.formats.FormatReader.setId(FormatReader.java:1392)
at loci.plugins.in.ImportProcess.initializeFile(ImportProcess.java:499)
at loci.plugins.in.ImportProcess.execute(ImportProcess.java:142)
at loci.plugins.in.Importer.showDialogs(Importer.java:140)
at loci.plugins.in.Importer.run(Importer.java:76)
at loci.plugins.LociImporter.run(LociImporter.java:78)
at ij.IJ.runUserPlugIn(IJ.java:235)
at ij.IJ.runPlugIn(IJ.java:198)
at ij.Executer.runCommand(Executer.java:150)
at ij.Executer.run(Executer.java:68)
at java.lang.Thread.run(Thread.java:748)
Caused by: java.lang.ClassNotFoundException: org.joda.time.ReadableInstant
at java.net.URLClassLoader.findClass(URLClassLoader.java:381)
at java.lang.ClassLoader.loadClass(ClassLoader.java:424)
at sun.misc.Launcher$AppClassLoader.loadClass(Launcher.java:349)
at java.lang.ClassLoader.loadClass(ClassLoader.java:357)
… 14 more

It saids that “file is not in asupported format, a reader plugin is not available, or it was not found” in opener window.

Yours Sincerely,
NaN