Fwd: Need trouble shooting advice for deconvolution

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Fwd: Need trouble shooting advice for deconvolution

Daniel James White
 Dear Dave,

Begin forwarded message:

> Date:    Sat, 12 Jun 2010 11:01:51 -0700
> From:    daschneider9 <[hidden email]>
> Subject: Need trouble shooting advice for deconvolution
>
> For those interested in deconvolution and willing to give advice to a
> non-math image processing novice:
>
> The goal is to deconvolve image stacks to improve subsequent 3D projections
> and colocalization analyses.

both things are a good idea! Its a good start already.

>
> My process:
> 1. specimen = ~4 um FFPE section of parasite-infected cultured cell, IFA
> labeled using Alexa488, mounted in slow fade gold

Good.

> 2. Slide for defining PSF = PS-Speck (Invitrogen) 0.17 um bead appropriate
> for "green" fluorescence, used mounting media provided (no specifics)

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.

Invotrogen tetraspk slides are mounted in optical galss glue stuff with eh RI of about the saem as glass.
Glycerol based mountants have a RI around 1.45 ish
Water is about 1.34.

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.

Ay High NA lens work, these little details make a dig difference to imaging and deconv results.

> 3. all imaging done the same way as follows: Zeiss upright, MrM camera using
> 256x256 window, 63x oil (NA-1.4), Zeiss immersion oil, exposure set to avoid
> saturation, 41 slices at 0.25 um spacing to allow additional capture
> out-of-focus light on either side of specimen focal plane

good.
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?


> 4. zvi stack files exported to tif series using export options "convert to 8
> bit" and "apply display mappings".

No No Noooooo!!!!!!!!!!!

open the zvi file directly using the LOCI bio-formats importer
-plugins-LOCI-bio-formats importer.

By converting to 8 bit tiff you threw away lots of info that is very useful for the coloc analysis to come.
The high dynamic range and good signal to noise are very much worth preserving!!!

THe display mapping might cause clipping of the top and bottom of the dynamic range of the images,
which is a total disaster for deconvolution and coloc analysis.

for coloc analysis in Fiji / ImageJ see
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis

> 5. "specimen" and "PSF" tif series each imported into Fiji as image sequence
> using "sort names numerically" as only checked option.

no need, use the Bio-formats importer, as it also read in the correct meta data about pixel spacing and z spacing etc.
They will be opened as probably 16 bit images, so you might need to adjust the contrast/brightness to see something!

your bead image also being from a 12 bit? camera is also a vewry food thing.
THe psf should have very high S:N which the image you posted seems to have.

BUT. You must remove the background from the PSF image ,
as that will mess up the deconv.
THe background in the ewPSF image seems to be about 10,
so you can subtract 10 from the whole image using
Process - math - subtract.
you might also want to so sometinhg similar in the sample  image .


> 6. Deconvolve using Parallel Iterative Decon..3D Iterative Deconv.

make double sure that the xy and z dimensions are exactly the same in
the bead and specimen image.

>  
> 7. Below I have attached the results each saved as a tif stack named by
> deconv method used. In all I used the defualt settings and 5 iterations.

For some of the methods 5 iterations might not be nearly enough.
Read the docs carefully at
http://pacific.mpi-cbg.de/wiki/index.php/Parallel_Iterative_Deconvolution

to understand how the different methods work and what oi might reasonably expect them to do well/badly .

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....

> 8. When this part is successful my next step is to perform colocalization
> analysis with a second deconvolved image of an Alexa555-taged probe.

see abiove link,
and feel free to as me about that too.

A red green merge image, and looking for yellow spots is a disaster,
you can measure something quite easily instead ands get numbers that can be compared with other numbers.
>
> WPL seems closest to an expected image except that, as in this example,
> frequently I loose distinction between individual parasites. Individual
> colonies seem better appreciated in the other methods but each of these
> seems to add its own "background" or artifacts that seem largely determined
> by pre-filter settings.

please try to post images that were not 8 bit.
these images are really badly intensity saturated/clipped

>
> Can anyone suggest where I'm going wrong here?
hopefully its clear from the above.

> Or what to try next in
> trouble shooting? I continue to read various websites and articles that
> provide instruction (mostly theory) but as yet I have not come up with a
> solution. I have had past success when I had trial access to the Zeiss
> Axiovision software for deconvolution (used its theoretical PSF generator)
> and also when I had trial access to another commericial program that used
> blind deconvolution. I mention this only to suggest that I don't think there
> is an inherent problem in the imaging setup.

In my hands the parallel iterative deconv plugin works well,
if you are careful and think about what food you give it,
and understand what is going on.

>
> All suggestions and advice are welcome. I am glad to provide any additional
> information requested.

hope  I helped,
fee free to ask more.

cheers

Dan



>
> Many thanks,
>
> Dave
>
> Images:
> http://imagej.588099.n2.nabble.com/file/n5172219/MRNSD.tif MRNSD.tif
> http://imagej.588099.n2.nabble.com/file/n5172219/WPL.tif WPL.tif
> http://imagej.588099.n2.nabble.com/file/n5172219/CGLS.tif CGLS.tif
> http://imagej.588099.n2.nabble.com/file/n5172219/HyBR.tif HyBR.tif
> --
> View this message in context: http://imagej.588099.n2.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp5172219p5172219.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> ------------------------------
>
> Date:    Sat, 12 Jun 2010 11:18:05 -0700
> From:    daschneider9 <[hidden email]>
> Subject: Re: Need trouble shooting advice for deconvolution
>
> It occurs to me that those interested in helping might want to see the PSF I
> created. Here it is:
> http://imagej.588099.n2.nabble.com/file/n5172275/PS-Speck-green.tif
> PS-Speck-green.tif
>
> Thanks again to those who reply,
> Dave
> --
> View this message in context: http://imagej.588099.n2.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp5172219p5172275.html
> Sent from the ImageJ mailing list archive at Nabble.com.

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )
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Re: Fwd: Need trouble shooting advice for deconvolution

daschneider9
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