[Neuroscience] Re: Automatic focus control for microscope?

polly jo via neur-sci%40net.bio.net (by jopower from yahoo.com)
Wed May 9 15:08:15 EST 2007


--- tomte <tehgabriel from web.de> wrote:

> Hi!
> 
> Thanks for your reply.
> 
> > I think you may be mistaken about what is needed. 
> There is no reason
> > for a microscope to go out of focus unless there
> is some really weird
> > temperature change that is causing expansion and
> contraction.  High
> > quality microscopes are very stable and don't
> change position so once
> > you put an item into focus it stays in focus.
> 
> Well my microscope does. And as far as i know, there
> are many
> colleagues complaining about small drifts in their
> optics during
> fluorescence imaging.
> I guess that's why some companies came up with these
> autofocus systems
> using image processing software. But these are not
> suitable for
> fluorescence imaging since they require a continuous
> image. But i need
> to avoid this in order to protect my preparation
> from bleaching.
> 
> > Do you have particular trouble focusing or keeping
> focus?  Describe
> > your problem and perhaps we can help you solve it.
> 
> I am using an Olympus  IX-71 with a 40x objective
> for fluorescence
> imaging on cultures of hippocampal neurons.
> During a specific experiment i want to sample 10
> pictures with a time
> interval of 4 min between each. But sometimes (~50
> %) i can observe
> over the timecourse ot the experiment that the
> images loose focus
> (interstingly, i never see this with DIC).


HI,

just wondering if what you are seeing as drift is in
fact bleaching  ..... my experience is that regular UV
bleaches the fluorophore rather quickly especially in
live imaging....especially, since your DIc does'nt
show any change.
Would that make sense?

polly
> 
> 
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