[OpenSPIM] Some early images

[Pavel Tomancak]

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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