[Neuroscience] Re: Automatic focus control for microscope?

r norman via neur-sci%40net.bio.net (by r_s_norman from _comcast.net)
Wed May 9 12:51:05 EST 2007


On 9 May 2007 01:12:01 -0700, 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).
>

If the DIC is on the same Olympus microscope, then you can't blame it
on movement of the microscope!  Although it is possible that the
system does shift as far.  There will simply be another part of the
field in focus for the DIC image but the specific fluorescent
structure you have imaged on might be shallow enough to be out of the
field of focus.  Do you have a stable temperature environment?  Forty
minutes is perhaps a long enough interval for changes in room
temperature to have an effect.  Or use the focus stabilization systems
with visible light between your UV flashes.







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