http://imagej.273.s1.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp3687551p3687553.html
major changes to the Bio-Formats Importer. With the latest version, are you
> >
> > Date: Mon, 14 Jun 2010 15:15:06 -0700
> > From: daschneider9 <
[hidden email]>
> > Subject: Re: Need trouble shooting advice for deconvolution
> >
> > Piotr,
> >
> > Thank you for your suggesting. The original zvi file stores each channel
> as
> > 16 bit grey scale. Am I correct that I should not rescale these to 32 bit
> > befoer deconvolution?
>
> you dont need to do it manually.
>
> Your CCD camera is probably 12 bit .... (you should know that)
> so it counts up to 4095.
> So long as there are no 4095 pixels in the image stack then image is not
> saturated.
> Also, since imageJ/Fiji will open them as 16 bit images,
> you have all the numbers from 4095 to 65000 ish to grow up into during the
> contrast enhancement of deconvolution.
> In fact, as Piotr describes, it actually done in 32 bit float, with masses
> of dynamic range and precision.
>
> In the end, you need to make sure you to the auto contrast on the result
> just ot make sure it is displayed within the range of your display (which is
> only 8 bit)
> You might notice that the image seems to be rather dark at the end...
> dont worry, thats because the contrast is now so huge, that the bright
> objets are so much brighter than the dimmer ones,
> that your eyes cant see the dim ones from black. Here is where the Fire LUT
> comes in handy.
> There is much more dynamic range in the result than your display can show
> and your eyes can see with greyscale LUT.
>
> >
> > Dave
> > --
> > View this message in context:
>
http://imagej.588099.n2.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp5172219p5179557.html> > Sent from the ImageJ mailing list archive at Nabble.com.
> > On Jun 15, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:
> >
> >
> > Date: Mon, 14 Jun 2010 17:11:13 -0700
> > From: daschneider9 <
[hidden email]>
> > Subject: Re: Fwd: Need trouble shooting advice for deconvolution
> >
> > 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?
>
> If
>
> 1) the refractive indexed are carefully matched (sample and immersion oil)
> 2) the coverglass is the correct thickness
> (high NA oil and W lenses are designed for exactly 0.17mm!
> normal number 1.5 glasses are anywhere from 0.16-0.19
> and vary greatly from batch to batch. You need the more expensive
> high precision ones from Zeiss, or Hecht Assistent etc. )
> 3) the lens has not been dropped or scratched
>
> then there should be 0 spherical aberration and the PSF will be very close
> to symmetrical in Z.
>
> The DeltaVision microscope system comes with a range of immersion oils with
> different RI
> so you can correct for any spherical aberration in your system by using the
> right RI oil.
> Nice trick.
>
> For non dehydrated / watery samples,
> I suggest a highly corrected Water immersion lens as made my Zeiss and
> olymopus and others.
> eg 60x 1.2 Water Korr.
> The effective NA of the lens is limited by the RI of the sample,
> so if it is watery (RI 1.34) then having a NA 1.4 oil imersion lens in no
> help.
>
> If your samples are thick, then the spherical abberation problem gets worse
> and worse with depth in Z,
> so a water immersion lens for a watery sample is an even better idea.
>
> For a thick sample mounted in Glycerol ish medium, a glycerol immersion
> lens would be nice.
>
> >
> > The slide, coverglass, z-step distance, image dimension were all the same
> > for specimen and PSF images.
>
> If you used normal 1.5 coverglasses (please tell me they were not number
> 1!!!!!)
> then they might be close to beng the same.... but they miught not be, see
> above.
> For high resolution microscopy you must use the high precision coverslides.
>
> >
> > 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
>
> nope, dry the beads on to the coverglass, not the slide.
>
> > and then
> > used the provided coverslip media.
>
> no good really, unless you know its refractive index.
> mowiol/Dabco for dead dehydrated samples works but it can shrink the sample
> and distort it.
> Modern glycerol non hard setting media are great,
> eg prolong gold , etc.
>
> > 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,...
>
>
> that a very good idea.
>
> >
> >
> > 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?
>
> Always the coverslip.... if you dry them on.
> Else you can use a hard setting medium (for the sample aswell)
> and suspend the beads in that,
> so a bead of interest might be about the same distance from the coverslide
> as the sample is .
>
> >
> >
> > 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,
>
> thats OK
>
> > but not the z-spacing.
>
> Good.
>
> Actually, if you like, one can oversample the bead image to get a even
> better PSF with more high frequency info in it,
> (say 40nm in xy and 100 nm in z... so long as there is no bleaching)
> but then you really need to resample it down to the voxel spacing of the
> sample image for the deconvolution....
> so maybe no point in this case.
>
> > I always center the best focal
> > plane of the bead within the stack used for deconv (presuming that is
> best).
>
> the PSF must be exactly centered!!!!
> In X Y and Z or else you will generate garbage.
>
> The Fourier transforms in the methods will be wrong if the PSF is not
> perfectly centered.
>
> >
> >
> > 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)
>
> Ummm, yeah, that can happen with zvi file currently with bio-fortmats... i
> think they know about it,
> but you could send the bio-foromats team a sample image that behaves badly.
>
> >
> > 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?
>
> Nope, only if ou want it to be Displayed nicer on the screen.
>
> > I tested with and without doing this and see no
> > apparent difference.
> >
> indeed it should make no 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.
>
> deciding the background in the bead image is rather easy.
> But in the sample its harder.
> You can take a background image of an area in the sample where there are no
> objects
> using the same filter and CCD exposure time, and use that mean value.
>
> Donr use a rolling ball or other hard threshold.
> What you want to remove is the +ve offset and dark current background of
> the ccd camera.
> But not remove any real fluorescence intensity.
>
> >
> >
> > 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.
> >
>
> that means that the PSF is very wrong and/or the imput image data is
> intensity clipped/saturated.
>
> >
> > 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.
>
> You are by no means the only one who made that mistake.
> I spend a lot of time trying to convince people to think of digital images
> as just a bunch of numbers.... not analogue art
> (I'm really a spectroscopist you see......)
> the fisrt thing i always do when i open an image is immediately look at the
> intensity histogram (analyse - histogram)
> in log scale for fluorescence, as only in that representation can you
> really see intensity clipping and bad 0 offsets!!!
>
> > However, I
> > still get the "oversaturated" appearance of the 16 bit images imported
> from
> > zvi using LOCI and after first subtracting background.
>
> before or after deconv.
> Go to the middle of the stack where there is stuff,
> and do the auto brightness/contrast there.
>
> > Separation of the
> > different organisms is better than before, but are still appearing to be
> > nearly saturated after deconv.
>
> it might just be the display that needs adjusting.
>
> > 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.
>
> try and get your system to have as little spherical aberation as possible,
> it greatly improves resolution, esp in z, and also strongly increases
> contrast.
> >
> > 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.
>
> there are a bunch of file share sites, but they are mostly annoying....
> I have a drop box account....
>
> >
> > 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.
>
> any questions, just yell.
> >
> > I look forward to more questions/suggestions. I really want to learn how
> to
> > do this appropriately.
>
> So do I, and I am also still learning!
>
>
> cheers
>
> Damn
>
>
> >
> > Dave
> > --
> > View this message in context:
>
http://imagej.588099.n2.nabble.com/Fwd-Need-trouble-shooting-advice-for-deconvolution-tp5177845p5179893.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 )
>