Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6723656.html
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This is a bit over the top. Paper does not give a specular reflection,
it is just illuminated by the laser. That is most unlikely to be
hazardous (except that you might set the paper on fire). And 3W is more
than you are likely to get - our old Verdi-Mira would give about 700mW
at peak, and if you tuned it to 700nm so that you could see it the power
was far less than this. What you need to be careful about is glass
elements which can give a specular reflection. In the end there is no
substitute for common sense.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]]
On Behalf Of Zac Arrac Atelaz
Sent: Thursday, 25 August 2011 2:06 PM
To:
[hidden email]
Subject: Re: Uniformity of 2-photon illumination?
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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
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:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > *****
> >
> > 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:
http://confocal-microscopy-list.588098.n2.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6719119.html
> > Sent from the Confocal Microscopy List mailing list archive at
Nabble.com.
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