Posted by
daschneider9 on
Jun 12, 2010; 6:01pm
URL: http://imagej.273.s1.nabble.com/Need-trouble-shooting-advice-for-deconvolution-tp3687551.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:
MRNSD.tifWPL.tifCGLS.tifHyBR.tif