[OpenSPIM] Multiview Reconstruction- optimizing settings?

[Maarten Hilbrant]

Dear all,

nice to see some questions on the mailing list lately regarding Stephan Preibisch' multview plugin. After attending the EMBO workshop in Dresden in 2014,  I've been using the plugin for two years now and it is an essential part of our light sheet workflow. In a nutshell, we image insect embryos, embedded in agarose containing sub-resolution beads, using single-sided illumination with 8 or 16 angles per time point. In order to infer the biological relevance of RNAi phenotypes, we tend to look at many different specimens for few time points, rather than collecting time-lapse data for a small number of specimens. In practice, this means a lot of reconstructing and adjusting settings, although I find that, for most of our datasets, default settings work nicely for bead detection, registration and deconvolution ("Nicely" in this case meaning a significant increase in local contrast). Sometimes, however, registration is sub-optimal (and who knows, maybe it is also for the analyses that I normally quality as "nice''-). In these cases, as a first hint, inspection with the BigDataViewer reveals that the psf's of the different angles don't overlap properly.

- Does this sound familiar to other users of the plugin?
- What reconstruction parameters do you generally tinker with first?
- for example: do you spend a lot of time on bead detection (and subsequent exclusion)?
- Do you sometimes reject datasets for multi view reconstruction and if so, on what grounds? For example, I like to image "a lot" of agarose surrounding the embryo in order to have a large set of beads for reconstruction, but am not always very systematic about this. Is this crucial in your data acquisition?

any ideas appreciated, basically to make better use of this great tool.

cheers,
Maarten

******************************
Dr. Maarten Hilbrant
Postdoctoral Researcher
Zoological Institute
University of Cologne
Cologne Biocenter
Zülpicher Str. 47b
50674 Cologne
Germany
+49(0)221-4703238
m.hilbrant at uni-koeln.de

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