Posted by
Armstrong, Brian on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6724897.html
Hi Stuart, in short, I align the 2P beam just as described by Julio in previous e-mail reply. I suggest you call your Zeiss service rep and then watch how he aligns the 2P to the vis laser(s). Once you've seen it, the protocol should be very easy to replicate.
You may already have a saved configuration called "2P Align" in C:AIM/settings that you can use. Also, you should have received a mirror slide with a grid on it that is perfect for this kind of alignment.
Adjusting the beam path as Julio described is quite easy to do.
Also, don't look into the laser light with your remaining good eye (first heard this sound advice from George McNamara).
Cheers,
Brian D Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx-----Original Message-----
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Sent: Thursday, August 25, 2011 3:38 AM
To:
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Subject: Re: Uniformity of 2-photon illumination?
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Thank you very much for your replies.
Just to clear up a few points, I always
take Z sections of the slide just to make sure it wasn’t a case that the slide
was at an angle, (The system is not designed to hold slides so its always a bit
“Heath Robison” trying to get a slide underneath it!)
I had checked various sites around the
slide and I was getting consistent results, yesterday I scanned the slide with
the descanned detectors and got a similar image.
We want all the power we can get as would
love to be able to image more deeply into cardiac muscle, (is it a fair
assumption that more power will allow us to image deeper?) for that reason we
have a Coherent Chameleon Ultra II, and I believe its peak power is around
5.5W. However would the alignment not take place “down stream” of the OPO? So I
could just set that to its minimum intensity setting.
Anyway it is kind of irrelevant because the
system is under service contract with Zeiss, So all I need to do it get on the
phone and start whining at them, Although I would love to give laser alignment a
go myself!
Once again thank you for all your replies
Yours
Stuart McIntyre
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