Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6721067.html
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We align our 2-P laser every few weeks or months, as needed. If your system is similar to ours, you should have your 2-P bean steered from the laser to the scanner through two mirrors, one near the laser beam source, and one near the scanner. If you have a fiber, then the following does not apply. If you remove the protective plastic covers over the mirros, you will see the mirrors, along with their adjustment screws. The way we do it is to run a continuous scan with a fluorescent slide, and then slightly tweak the adjustment screws, optimizing for intensity and uniformity of the image on the screen. You can use linescan / line profile if you want to be more precise. Start with the adjustment screws near the scanner. You should be careful to do very small adjustments, so that you don't loose the laser beam completely, because then it gets a bit more complicated, but if you just tweak each screw one way and the other, and repeat a few times for all the screws, you should be able to get good uniform illumination. This works if the laser is pretty aligned overall, but just needs a little adjustment (which seems to be your case). Attenuate your laser before you do this (you should need less than 10% to get an image), wear protective glasses, and watch out for reflective objects (jewelry, etc, ) that might bounce the laser beam into your retina. Put warning signs, and/or keep people out of the room while you are doing this.
If you can't get bright, even illumination with this method, you may need more serious intervention. One trick we use is to use a visible laser, such as the 488 line, as a reference for what the ideal light path should be. Use a mirror slide to bounce the 488 back into the objective and along the 2-P light path (I guess you would need an 80/20 dichroic, so that some of the 488 power goes through). You can see the 488 beam with a piece of paper as it follows the 2-P path. The trick then is to get the 2-P beam to follow the same path, starting near the scanner and working your way back to the 2-P laser source. This is quite tricky, and it's quite easy to make things worse, so I don't recommend it unless you know your 2P beam is way off. I used this method once when our 2P beam had been completely lost and we couldn't even get an image on the screen. I did this long ago, so I can't guarantee all the details, but you should get the idea. Calling a service person might also be a good alternative in this case.
Again, be very careful. I once burned a hole through a piece of paper (and burned my finger), because I had the 2P at high power and was being a bit casual with it. Burning your finger is not a big deal (it will hurt before you do serious damage to it), but your eye (or someone else's) is a different story...
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
http://www.fhcrc.orgOn Aug 24, 2011, at 12:14 AM, stu_the_flat wrote:
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> Dear list users,
>
> We have a Zeiss 510 two-photon system, it seems to have a an non-uniform
> field of illumination,
>
> I have tried to test this by imaging a chroma fluorescent test slide there
> is a two fold drop in fluorescent signal across the X axis, the there
> appears to be a slight variation on the Y axis as well,
>
> What is most likely to be causing this? Is imaging a test slide a fair test?
>
> Any advice would be most welcome.
>
> Thank you
>
> Stuart McIntyre
>
>
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