[OpenSPIM] Some early images

[Michael Weber]

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> 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> 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
> 
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_____________

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

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