Posted by
daschneider9 on
Jun 15, 2010; 12:11am
URL: http://imagej.273.s1.nabble.com/Fwd-Need-trouble-shooting-advice-for-deconvolution-tp3687923p3687924.html
Dan,
Thank you. I'll try to respond to all your questions:
Daniel James White wrote
The bead image shows there is quote bad spherical aberration. The PSF is not symmetrical in Z. If the bead is mounted in different stuff than the sample is, and the sample is more than a micron or 2 away from the coverglass, then it might not be the right PSF for deconvolving the image stack, as the spherical aberration will not be the same.
Is this z-asymetry not typical of widefield or is it particularly bad in my image stack?
The slide, coverglass, z-step distance, image dimension were all the same for specimen and PSF images.
The PS-Speck slide used to create this PSF image was made by me as described by the manufacturer. Basically dried diluted PS-Speck onto a
slide and then used the provided coverslip media. I too wondered about the potential for different indices for the two mounting mediums used. I will make a new PS-Speck slide with the mounting media used on the specimen slide. However,...
Daniel James White wrote
You must make sure the coverslip thickness is exactly the same for the bead sample and the actual sample, else again you will get different spherical aberration of the PSF.
So is it best to dry the PS-Specks onto the slide (which is how the 5-um specimen sections were processed) or onto the coverslip?
Daniel James White wrote
But are the pixel spacings the same for the real image stack and the bead stack? Same optovar? Same Camera, same binning? Same z-spacing?
Yes. All of these details are the same. Sometimes the number of slices captured is different, but not the z-spacing. I always center the best focal plane of the bead within the stack used for deconv (presuming that is best).
Daniel James White wrote
open the zvi file directly using the LOCI bio-formats importer-plugins-LOCI-bio-formats importer.
I can do that. However I get the following exception log after opening using the separate channels option only:
java.lang.NullPointerException
at ij.gui.ImageWindow.close(ImageWindow.java:371)
at ij.gui.StackWindow.close(StackWindow.java:139)
at ij.ImagePlus.close(ImagePlus.java:331)
at loci.plugins.importer.Importer.displayStack(Importer.java:691)
at loci.plugins.importer.Importer.showStack(Importer.java:559)
at loci.plugins.importer.Importer.run(Importer.java:401)
at loci.plugins.LociImporter.run(LociImporter.java:77)
at ij.IJ.runUserPlugIn(IJ.java:189)
at ij.IJ.runPlugIn(IJ.java:155)
at ij.Executer.runCommand(Executer.java:147)
at ij.Executer.run(Executer.java:78)
at java.lang.Thread.run(Thread.java:619)The images seem fine despite this message. They are 16 bit as expected and not B/C adjusted. Do I need to do B/C adjustment (pressing auto and then apply) before deconvolution? I tested with and without doing this and see no apparent difference.
Daniel James White wrote
...you can subtract 10 from the whole image using Process - math - subtract.
you might also want to so sometinhg similar in the sample image .
Yes, this does improve the deconv image some. Slightly better when the same is done to specimen image.
Daniel James White wrote
For some of the methods 5 iterations might not be nearly enough.
I agree but the abberations only get worse with more interation and I was just trying to show how badly things were right off the starting block.
Daniel James White wrote
the images you posted are all very bady saturated.... this is because you made the 8 bit, and then im bot sure the deconvolution handles 8 bit images properly....you needs 16 bit ot even 32 bit fload images for the deconv to not run out of headroom. Deconv will greatly increase the dynamic range of the image....
Yes, the 8 bit conversion is a mistake I should not have made and I thank you and Poitr for pointing this out--A rooky mistake for sure. However, I still get the "oversaturated" appearance of the 16 bit images imported from zvi using LOCI and after first subtracting background. Separation of the different organisms is better than before, but are still appearing to be nearly saturated after deconv. Am I still missing something else? Perhaps there is still too many problems as yet with my PSF. I'll get on making new PSF files and try again.
I'd be happy to post the original zvi files (~16 MB each) but nabble is refusing them (too big is the reply). Is there another site people are using for posting such image links? I will also provide them directly to any who would like to help.
I can't thank you enough for your time. I really apprecaite the linked resources as well. I've read them a few times but obviously have to keep re-reading them to see what details I'm not picking up on.
I look forward to more questions/suggestions. I really want to learn how to do this appropriately.
Dave