http://confocal-microscopy-list.275.s1.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6724541.html
Sorry all. I wrote what I did with an assumption, and that's not good. I
something as powerful as a 2P laser. When I used a card, I had the ability
the laser spot was at an extremely low power level. I did this for years,
probably as often as 3 times a week. I expect that this was not an
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>
> Laser users:
>
> Please, anyone trying to align lasers, consider first
>
http://en.wikipedia.org/wiki/Laser_safety this is basic, in all the older
> 2-P systems you have the highest classification of danger from a laser,
> that is class IV!!! even reflection from paper can be terribly dangerous,
> when installing those systems a interlock is a must so if anyone opens the
> door while working, the laser beam is blocked or shut down, you will notice
> that the peak power of the system is almost 3W, but when pulsed this can go
> over the roof, the peak power can reach in some models up to 380,000W, 10%
> of that is quite a bunch.
>
> So if you remember that paper reflectance can go from 50 to 90%, you can
> picture that its not safe at all putting a piece of paper following laser
> paths, as you can get half or almost all the laser going in unknown angles
> around the room
>
>
http://www.laserfx.com/BasicSafety/BasicSafety2.html>
> Going trough numbers we have this:
>
> Safe exposure = 2.5mW /cm2
> 1W trough your eye = 100,000W /cm2
>
> Placing there the piece of paper in the way you will still have half of
> this energy going in the angle that your hand is giving to the paper, with
> the pulsed laser you will have a quadrillion of laser hits by second
>
> So believe me is safer and easier having the interlock activated, and being
> reaaaally careful about this devices, by the way there is only one 2-P or MP
> microscope in the market that after installed goes down in laser safety
> requirements, you can be trained to align lasers, but is a really precise
> and dangerous task.
>
> Best regards
>
> Gabriel OH
>
>
>
>
>
> > Date: Wed, 24 Aug 2011 10:20:20 -0700
> > From:
[hidden email]
> > Subject: Re: Uniformity of 2-photon illumination?
> > To:
[hidden email]
> >
> > *****
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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.org> >
> >
> >
> > On Aug 24, 2011, at 12:14 AM, stu_the_flat wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
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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
> > >
> > >
> > > --
> > > View this message in context:
>
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