http://imagej.273.s1.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp3687551p3687556.html
> 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.
>
> My process:
> 1. specimen = ~4 um FFPE section of parasite-infected cultured cell, IFA
> labeled using Alexa488, mounted in slow fade gold
> 2. Slide for defining PSF = PS-Speck (Invitrogen) 0.17 um bead appropriate
> for "green" fluorescence, used mounting media provided (no specifics)
> 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
> 4. zvi stack files exported to tif series using export options "convert to 8
> bit" and "apply display mappings".
> 5. "specimen" and "PSF" tif series each imported into Fiji as image sequence
> using "sort names numerically" as only checked option.
> 6. Deconvolve using Parallel Iterative Decon..3D Iterative Deconv.
> 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.
> 8. When this part is successful my next step is to perform colocalization
> analysis with a second deconvolved image of an Alexa555-taged probe.
>
> 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.
>
> Can anyone suggest where I'm going wrong here? 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.
>
> All suggestions and advice are welcome. I am glad to provide any additional
> information requested.
>
> 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
> --
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