[OpenSPIM] Some early images

[Kevin W Eliceiri]

great, thanks Michael, Julie please order some of these.

best
k

On 06/17/13, Michael Weber  wrote:
> 
> 
> 
> Hi everyone,
> 
> 
> 20 um beads are way too big for this purpose and result in the super-bright balls which oversaturate the CCD. We have good experience with the 0.5 um Estaphor "green" beads (well vortexed, try w/o sample first!). I added a page "beads" to the wiki and linked it to the sample preparation section:
> 
> 
> http://openspim.org/Sample_Preparation
> 
> 
> So far you find some information about the beads I used in Pavel's and Jan's lab. I suggest to move bead-relevant stuff on this one page, rather than spreading it to individual pages.
> 
> 
> cheers,
> Michael
> 
> 
> 
> On Jun 14, 2013, at 8:41 PM, Pavel Tomancak <tomancak at mpi-cbg.de <tomancak at mpi-cbg.de>> wrote:
> 
> 
> > I will expand the section 
> > 
> > http://openspim.org/Drosophila_embryo_sample_preparation#What_beads_to_use.3F
> > 
> > 
> > on the bead selection. It is a complex problem with three variables, the strength of your signal, the laser power and the properties of your emission filter (band-pass versus long-pass). I recommend to get several wavelengths of beads and experiment with your particular conditions. I can provide some guidelines for His-YFP.
> > 
> > 
> > That the lines come from beads came as a surprise to me. This is most likely caused by the fact that they are too large, i.e. not sub resolution. Bright beads can be a little disturbing but I have never seen anything like that.
> > 
> > 
> > All the best
> > 
> > 
> > PAvel
> > 
> > 
> > On Jun 14, 2013, at 6:22 PM, Johannes Schindelin <Johannes.Schindelin at gmx.de <Johannes.Schindelin at gmx.de>> wrote:
> > 
> > 
> > > Hi Luke,
> > > 
> > > On Fri, 14 Jun 2013, Luke Stuyvenberg wrote:
> > > 
> > > 
> > > > On 06/14/13, Johannes Schindelin wrote:
> > > > 
> > > > 
> > > > > Also, as I told Julie on her way out: we absolutely have to use
> > > > > off-frequency beads. Our signal is too delicate to be overlaid by
> > > > > beads having a field day.
> > > > > 
> > > > 
> > > > I agree. Before we finished imaging, Julie decided to work with the red
> > > > beads on Monday; next Thursday we should be able to put together some
> > > > samples with weaker beads.
> > > > 
> > > > 
> > > > > This might need some changes on the Wiki to make it *very clear* that
> > > > > you should *never* use beads that are excited by exactly the
> > > > > wavelength of the laser.
> > > > > 
> > > > 
> > > > I should point out that these are 20um beads causing the flares. It's
> > > > possible that sub-resolution beads (even those specifically excited by
> > > > our laser) won't have the same dramatic effect as these. In other words
> > > > '*never*' might be a bit overzealous; I'm sure there are times when
> > > > operators would like a very strong bead signal.
> > > > 
> > > 
> > > Given that Dresden had substantial problems even with sub-resolution
> > > beads, I would actually wager a bet that "never" is the correct adjective
> > > here: I highly doubt that any non-toxic amounts of fluorophores translated
> > > from reporter genes would come even close to the amount of fluorophores
> > > present even in the tiniest sub-resolution beads made by humans.
> > > 
> > > In any case, the idea of OpenSPIM is to make things easier by sharing
> > > knowledge, right? So even if I'd lose my bet, it would be the appropriate
> > > thing to share the insight that non-off-color beads *were* too bright in
> > > our case.
> > > 
> > > Ciao,
> > > Johannes
> > 
> > 
> > 
> > 
> > _______________________________________________
> > OpenSPIM mailing list
> > OpenSPIM at openspim.org <OpenSPIM at openspim.org>
> > http://openspim.org/mailman/listinfo/openspim
> > 
> 
> 
> _____________
> 
> Michael Weber
> PhD Student, Huisken lab
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108, 01307 Dresden
> Tel. 0049 351/2102837
> 
> http://www.mpi-cbg.de/huisken

--
Kevin W. Eliceiri
Director, Laboratory for Optical and Computational Instrumentation (LOCI)
Departments Cell and Molecular Biology and Biomedical Engineering
Affiliate Principal Investigator, Morgridge Institute for Research (MIR)
Room 271 Animal Sciences, 1675 Observatory Drive, Madison, WI 53706
Phone: 608-263-6288