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Uniformity of 2-photon illumination?

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stu_the_flat

Uniformity of 2-photon illumination?

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

Vladimir Ghukasyan-2

Re: Uniformity of 2-photon illumination?

*****
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Stuart,

Most probably you see a result of a bad aligned laser beam in the scanning unit.

Best wishes,
Vladimir

On Wed, Aug 24, 2011 at 3:14 AM, stu_the_flat <[hidden email]> 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.
>

Guy Cox-2

Re: Uniformity of 2-photon illumination?

In reply to this post by stu_the_flat

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The Chroma slide should be a pretty good test. You should do 2 or 3
focal positions in case the effect is actually a tilt in the stage or
the optics. Do you see the same effect in the confocal and
non-descanned detectors? If so it's clearly not something interfering
with the detection optics, and you'd have to blame the illumination.

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 stu_the_flat
Sent: Wednesday, 24 August 2011 5:14 PM
To: [hidden email]
Subject: Uniformity of 2-photon illumination?

*****
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-pho
ton-illumination-tp6719119p6719119.html
Sent from the Confocal Microscopy List mailing list archive at
Nabble.com.

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1392 / Virus Database: 1520/3853 - Release Date: 08/23/11

Jerry (Gerald) Sedgewick

Re: Uniformity of 2-photon illumination?

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

To determine if the laser is not centered, you might want to do one of two
things that has worked for me. You can set the laser at a wavelength you
can see closer to 720nm. Place business card or the like under a 10X lens.
Activate the laser and then focus up and down. If the spot does not remain
centered as you move up and down, the laser is off axis with the optics. My
thought is that the laser would likely be badly misaligned in this scenario.

For another means of determining a misaligned laser, print a target with
larger and larger circles so that this can fit nicely over the field lens
(you will have to measure the diameter of the field lens and then make the
outermost circle at that diameter, cut out the target using the outermost
diameter as a guide, so that the target is centered). Use a 4x or 10x lens,
remove the condensor and activate laser. You should be able to see the
illumination pattern.

This target is a nice thing to use when aligning the laser yourself (if you
do not void your service agreement/contract). With 2 people in the room,
one aligning and the other looking at the target, the task of alignment is
made easier. While it's true that an attachment for the microscope can be
had for centering, the fact that head movement in relation to that device
always made me less certain about its efficacy.

Otherwise, like Guy said, the Chroma slide is a good test.

Jerry Sedgewick

On Wed, Aug 24, 2011 at 2:31 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The Chroma slide should be a pretty good test. You should do 2 or 3
> focal positions in case the effect is actually a tilt in the stage or
> the optics. Do you see the same effect in the confocal and
> non-descanned detectors? If so it's clearly not something interfering
> with the detection optics, and you'd have to blame the illumination.
>
> 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 stu_the_flat
> Sent: Wednesday, 24 August 2011 5:14 PM
> To: [hidden email]
> Subject: Uniformity of 2-photon illumination?
>
> *****
> 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-pho
> ton-illumination-tp6719119p6719119.html
> Sent from the Confocal Microscopy List mailing list archive at
> Nabble.com.
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1392 / Virus Database: 1520/3853 - Release Date: 08/23/11
>

Julio Vazquez

Re: Uniformity of 2-photon illumination?

In reply to this post by stu_the_flat

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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:

Zac Arrac Atelaz

Re: Uniformity of 2-photon illumination?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
> > 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.

Guy Cox-2

Re: Uniformity of 2-photon illumination?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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-pho
ton-illumination-tp6719119p6719119.html
> > Sent from the Confocal Microscopy List mailing list archive at
Nabble.com.

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1392 / Virus Database: 1520/3854 - Release Date: 08/24/11

stu_the_flat

Re: Uniformity of 2-photon illumination?

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

George McNamara

Re: Uniformity of 2-photon illumination?

In reply to this post by Guy Cox-2

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy,

A laminated business card may be a different story.

Coherent set up a Chameleon Ultra II Compact OPO in my lab for several
days in early August (thanks again to Scott Crane and Ricky Skillings!).
They were visiting because we are including an OPO-Vis in our Leica STED
grant proposal (the CW-STED demo is going nicely, except for CW's
limited STEDable colors, 5x advantage of pulsing over CW for depletion,
advantage of time gating by pulsed excitation, and ~2 hours if
vibrations some days - my core is on the 6th floor and the demo
vibration isolation table has skinny legs).

The Chameleon OPO peak (avg) power was 4.8 Watts at 800 nm. The Rimke
FOM 2011 abstract from APE and Coherent shows complete power curves for
the standard Chameleon OPO-Vis outputs
http://www.focusonmicroscopy.org/2011/PDF/422_Rimke.pdf (for extra
money the idler beam can also be exported ... this is ~1700 nm to 4000
nm, so not clear if it has any use or how we would get it into a useful
wavelength range).

Enjoy and be safe,

Sincerely,

George

On 8/25/2011 4:35 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> 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]
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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:
>>> 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-pho
> ton-illumination-tp6719119p6719119.html
>
>>> Sent from the Confocal Microscopy List mailing list archive at
>>>
> Nabble.com.
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 10.0.1392 / Virus Database: 1520/3854 - Release Date: 08/24/11
>
>

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

Jerry (Gerald) Sedgewick

Re: Uniformity of 2-photon illumination?

In reply to this post by Zac Arrac Atelaz

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Sorry all. I wrote what I did with an assumption, and that's not good. I
assumed that everyone would be careful/aware/safety conscious about
something as powerful as a 2P laser. When I used a card, I had the ability
to turn down the laser with a neutral density wheel to the level at which
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
appropriate recommendation/method except for those who build
custom 2photons, or those who service lasers.

Jerry Sedgewick

On Wed, Aug 24, 2011 at 11:06 PM, Zac Arrac Atelaz <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> 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]
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > 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:
> > > 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.
>

Armstrong, Brian

Re: Uniformity of 2-photon illumination?

In reply to this post by stu_the_flat

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-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of stu_the_flat
Sent: Thursday, August 25, 2011 3:38 AM
To: [hidden email]
Subject: Re: Uniformity of 2-photon illumination?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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

--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6723940.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

---------------------------------------------------------------------
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This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.

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Craig Brideau

Re: Uniformity of 2-photon illumination?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Just set your power control up as close to your laser as possible. I use a
1/2 waveplate and polarizing splitter cube with a sturdy metal beam dump.
That way any beam from that point onward is at most a few hundred mW, and
for most imaging applications 200mW or less is plenty. I also keep a few
movable beam blocks handy at all times. If I need to work with the laser, I
throw multiple blocks into the system, then remove them as I go. That way
the laser is never going further than the next block in the system. Here is
my blocking 'recipe':

For the actual blocks, I use Thorlabs blank cage plates (CP02)
http://www.thorlabs.com/thorProduct.cfm?partNumber=CP01
These can handle the full brunt of a Ti:Saph if necessary, and the
black anodizing tends to absorb the beam nicely.

I put the CP02 on a standard post TR3
http://www.thorlabs.com/thorProduct.cfm?partNumber=TR3

I put the post in a magnetic post holder PH3E
http://www.thorlabs.com/thorProduct.cfm?partNumber=PH3E

I usually use them in pairs; one blocks the laser a bit further down the
table, while the other I keep up close. The magnetic holder allows you to
easily move and reposition them. By using multiple blocks, I ensure there
is always at least one somewhere in the beam. I only remove the last one
when I am ready to shoot the beam into the microscope. The only caution I
have is the TR3 post is shiny stainless, so always move the blocks sideways
out of the beam rather than lifting them straight up. (you can paint the
exposed portion of them black if it is an issue) If you get PH4 holders you
can drop the post completely into the post holder, but this limits your
ability to move the block up and down. Collar R2 can help cover up the post
and be used to lock it to a specific height:

http://www.thorlabs.com/thorProduct.cfm?partNumber=R2

Hope this helps!

Craig

On Thu, Aug 25, 2011 at 9:36 AM, Armstrong, Brian <[hidden email]>wrote:

> 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-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of stu_the_flat
> Sent: Thursday, August 25, 2011 3:38 AM
> To: [hidden email]
> Subject: Re: Uniformity of 2-photon illumination?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
>
>
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/Uniformity-of-2-photon-illumination-tp6719119p6723940.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
> ---------------------------------------------------------------------
> *SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or
> entity to which they are addressed. This communication may contain
> information that is privileged, confidential, or exempt from disclosure
> under applicable law (e.g., personal health information, research data,
> financial information). Because this e-mail has been sent without
> encryption, individuals other than the intended recipient may be able to
> view the information, forward it to others or tamper with the information
> without the knowledge or consent of the sender. If you are not the intended
> recipient, or the employee or person responsible for delivering the message
> to the intended recipient, any dissemination, distribution or copying of the
> communication is strictly prohibited. If you received the communication in
> error, please notify the sender immediately by replying to this message and
> deleting the message and any accompanying files from your system. If, due to
> the security risks, you do not wish to receive further communications via
> e-mail, please reply to this message and inform the sender that you do not
> wish to receive further e-mail from the sender.
>
> ---------------------------------------------------------------------
>
>

Marco Marcello

Newport MaiTai 2-photon laser maintenance

In reply to this post by Guy Cox-2

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Colleagues,

Do you own/you want to buy a two photon laser from Spectra Physics/Newport?
I would like to share my experience with the Spectra Physics/Newport service :

15.03.2011 Our MaiTai laser has a very low output power and doesn't pulse. I contact the service engineer.

29.03.2011 After two weeks of mail exchange with the engineer trying to solve the problem in remote,
it turns out that this is something more serious. The engineer comes over, collects the laser and promise we will have the laser back in 1,5-2 months. End of May then. That is the only Multi Photon laser in the facility and I start already to have worried users around asking when the laser will be back.

03.06.2011 I write Spectra Physics (SP) urging news about the laser.

27.06.2011 SP informs me that they will start to work on the laser "this week" and the laser should be back in the week of the 11th of July. They added "If it is possible to do it any earlier we will".

01.07.2011 SP informs me that there are good news: the laser will be really back by the middle of July.

14.07.2011 No news from SP. I write them asking about an update. SP answers quickly: new installation date, the 3rd of August. 2011? My bad, I forgot to ask them.

05.08.2011 Again no news and no laser back. I write this time the Sales Director a complaint letter asking when we will have our laser back and if it is possible to have a replacement instrument, as we are without laser since more than 4 months now.

17.08.2011 Receiving no answer from the Sales Director I call him, and receive assurance that the laser will be back next week.

26.08.2011 Still nothing. I write a new complaint mail to the Sales Director. He answers that there were more delays, but by the 31st of August he will surely be able to know more about when the laser will be back and he will contact me.

01.09.2011 No news from SP (to be continued).

Now it might well be that all of it is absolutely normal, I just would like to get a feedback from the community if there is anything else I can do in order to have the laser back.
Thanks for reading this overly long post and for your feedback.

Cheers,

Marco

Marco Marcello
Imaging Manager
Centre for Cellular Imaging, IIB
University of Liverpool
UK

Sylvie Le Guyader-2

Re: Newport MaiTai 2-photon laser maintenance

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Ouch!

I guess the strong stand you are taking now is the obvious desperate next step!

Best of luck with it all!

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: +46 (0) 8 5248 1107
LCI room: +46 (0) 8 5248 1172
mobile: +46 (0) 73 733 5008

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of CCI
> Sent: 01 September 2011 13:17
> To: [hidden email]
> Subject: Newport MaiTai 2-photon laser maintenance
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> Do you own/you want to buy a two photon laser from Spectra Physics/Newport?
> I would like to share my experience with the Spectra Physics/Newport service :
>
> 15.03.2011 Our MaiTai laser has a very low output power and doesn't pulse. I
> contact the service engineer.
>
> 29.03.2011 After two weeks of mail exchange with the engineer trying to solve the
> problem in remote,
> it turns out that this is something more serious. The engineer comes over, collects
> the laser and promise we will have the laser back in 1,5-2 months. End of May
> then. That is the only Multi Photon laser in the facility and I start already to have
> worried users around asking when the laser will be back.
>
> 03.06.2011 I write Spectra Physics (SP) urging news about the laser.
>
> 27.06.2011 SP informs me that they will start to work on the laser "this week" and
> the laser should be back in the week of the 11th of July. They added "If it is
> possible to do it any earlier we will".
>
> 01.07.2011 SP informs me that there are good news: the laser will be really back by
> the middle of July.
>
> 14.07.2011 No news from SP. I write them asking about an update. SP answers
> quickly: new installation date, the 3rd of August. 2011? My bad, I forgot to ask
> them.
>
> 05.08.2011 Again no news and no laser back. I write this time the Sales Director a
> complaint letter asking when we will have our laser back and if it is possible to
> have a replacement instrument, as we are without laser since more than 4 months
> now.
>
> 17.08.2011 Receiving no answer from the Sales Director I call him, and receive
> assurance that the laser will be back next week.
>
> 26.08.2011 Still nothing. I write a new complaint mail to the Sales Director. He
> answers that there were more delays, but by the 31st of August he will surely be
> able to know more about when the laser will be back and he will contact me.
>
> 01.09.2011 No news from SP (to be continued).
>
> Now it might well be that all of it is absolutely normal, I just would like to get a
> feedback from the community if there is anything else I can do in order to have the
> laser back.
> Thanks for reading this overly long post and for your feedback.
>
> Cheers,
>
> Marco
>
>
> Marco Marcello
> Imaging Manager
> Centre for Cellular Imaging, IIB
> University of Liverpool
> UK

Neeraj Gohad-2

Re: digestion of cuticle

*****
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*****

Dear list,

Does anyone have a good protocol or know a reference for digesting arthropod cuticle with chitinase?

Thanks,

Neeraj.

Neeraj V. Gohad, Ph.D.
Research Assistant Professor
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: 864-656-3597
Fax: 864-656-0435

Sudipta Maiti

Re: Newport MaiTai 2-photon laser maintenance

In reply to this post by Marco Marcello

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We have had a MaiTai for the last >2 years, and it has been mostly down. Right
now it has been down for about three months, though it is under a maintenance
contract!
Sudipta
On Thu, 1 Sep 2011 11:16:53 +0000, CCI wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> Do you own/you want to buy a two photon laser from Spectra Physics/Newport?
> I would like to share my experience with the Spectra Physics/Newport
> service :
>
> 15.03.2011 Our MaiTai laser has a very low output power and doesn't
> pulse. I contact the service engineer.
>
> 29.03.2011 After two weeks of mail exchange with the engineer trying
> to solve the problem in remote, it turns out that this is something
> more serious. The engineer comes over, collects the laser and
> promise we will have the laser back in 1,5-2 months. End of May
> then. That is the only Multi Photon laser in the facility and I
> start already to have worried users around asking when the laser
> will be back.
>
> 03.06.2011 I write Spectra Physics (SP) urging news about the laser.
>
> 27.06.2011 SP informs me that they will start to work on the laser
> "this week" and the laser should be back in the week of the 11th of
> July. They added "If it is possible to do it any earlier we will".
>
> 01.07.2011 SP informs me that there are good news: the laser will be
> really back by the middle of July.
>
> 14.07.2011 No news from SP. I write them asking about an update. SP
> answers quickly: new installation date, the 3rd of August. 2011? My
> bad, I forgot to ask them.
>
> 05.08.2011 Again no news and no laser back. I write this time the
> Sales Director a complaint letter asking when we will have our laser
> back and if it is possible to have a replacement instrument, as we
> are without laser since more than 4 months now.
>
> 17.08.2011 Receiving no answer from the Sales Director I call him,
> and receive assurance that the laser will be back next week.
>
> 26.08.2011 Still nothing. I write a new complaint mail to the Sales
> Director. He answers that there were more delays, but by the 31st of
> August he will surely be able to know more about when the laser will
> be back and he will contact me.
>
> 01.09.2011 No news from SP (to be continued).
>
> Now it might well be that all of it is absolutely normal, I just
> would like to get a feedback from the community if there is
> anything else I can do in order to have the laser back. Thanks for
> reading this overly long post and for your feedback.
>
> Cheers,
>
> Marco
>
> Marco Marcello
> Imaging Manager
> Centre for Cellular Imaging, IIB
> University of Liverpool
> UK

Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
alternate e-mail: [hidden email]
url: www.biophotonics.in

Zac Arrac Atelaz

Re: Newport MaiTai 2-photon laser maintenance

In reply to this post by Marco Marcello

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*****

Marco:

This is the kind of really important and nice information to have before making choices, thank you so much for sharing!!!! I think this is not so long, I feel you were pushing your frustration back into the cage to make this so straight forward. I would like to ask what is the worked hours for this device before failure?, once again thank you

Gabriel OH

> Date: Thu, 1 Sep 2011 11:16:53 +0000
> From: [hidden email]
> Subject: Newport MaiTai 2-photon laser maintenance
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> Do you own/you want to buy a two photon laser from Spectra Physics/Newport?
> I would like to share my experience with the Spectra Physics/Newport service :
>
> 15.03.2011 Our MaiTai laser has a very low output power and doesn't pulse. I contact the service engineer.
>
> 29.03.2011 After two weeks of mail exchange with the engineer trying to solve the problem in remote,
> it turns out that this is something more serious. The engineer comes over, collects the laser and promise we will have the laser back in 1,5-2 months. End of May then. That is the only Multi Photon laser in the facility and I start already to have worried users around asking when the laser will be back.
>
> 03.06.2011 I write Spectra Physics (SP) urging news about the laser.
>
> 27.06.2011 SP informs me that they will start to work on the laser "this week" and the laser should be back in the week of the 11th of July. They added "If it is possible to do it any earlier we will".
>
> 01.07.2011 SP informs me that there are good news: the laser will be really back by the middle of July.
>
> 14.07.2011 No news from SP. I write them asking about an update. SP answers quickly: new installation date, the 3rd of August. 2011? My bad, I forgot to ask them.
>
> 05.08.2011 Again no news and no laser back. I write this time the Sales Director a complaint letter asking when we will have our laser back and if it is possible to have a replacement instrument, as we are without laser since more than 4 months now.
>
> 17.08.2011 Receiving no answer from the Sales Director I call him, and receive assurance that the laser will be back next week.
>
> 26.08.2011 Still nothing. I write a new complaint mail to the Sales Director. He answers that there were more delays, but by the 31st of August he will surely be able to know more about when the laser will be back and he will contact me.
>
> 01.09.2011 No news from SP (to be continued).
>
> Now it might well be that all of it is absolutely normal, I just would like to get a feedback from the community if there is anything else I can do in order to have the laser back.
> Thanks for reading this overly long post and for your feedback.
>
> Cheers,
>
> Marco
>
>
> Marco Marcello
> Imaging Manager
> Centre for Cellular Imaging, IIB
> University of Liverpool
> UK

Craig Brideau

Re: Newport MaiTai 2-photon laser maintenance

In reply to this post by Sylvie Le Guyader-2

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Service from any company seems to be very hit-and-miss these days. I have
noticed severe slowdowns whenever I need anything done from almost every
company. From what I have heard, support tends to be one of the first
things that gets cut when a company needs to downsize. As a result, all the
existing customers suffer. I keep hearing stories about how this or that
service rep or even manager has been laid off! The key thing is to find out
how service is in your local area with regards to whatever company you are
considering purchasing from. If you are unlucky, the service rep in your
area will either have been laid off, or is so badly overworked he'll never
have the time to get to you.'

Craig

On Thu, Sep 1, 2011 at 5:34 AM, Sylvie LeGuyader <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ouch!
>
> I guess the strong stand you are taking now is the obvious desperate next
> step!
>
> Best of luck with it all!
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader
> Live Cell Imaging Unit
> Dept of Biosciences and Nutrition
> Karolinska Institutet
> Novum
> 14183 Huddinge
> Sweden
> office: +46 (0) 8 5248 1107
> LCI room: +46 (0) 8 5248 1172
> mobile: +46 (0) 73 733 5008
>
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]] On Behalf Of CCI
> > Sent: 01 September 2011 13:17
> > To: [hidden email]
> > Subject: Newport MaiTai 2-photon laser maintenance
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear Colleagues,
> >
> > Do you own/you want to buy a two photon laser from Spectra
> Physics/Newport?
> > I would like to share my experience with the Spectra Physics/Newport
> service :
> >
> > 15.03.2011 Our MaiTai laser has a very low output power and doesn't
> pulse. I
> > contact the service engineer.
> >
> > 29.03.2011 After two weeks of mail exchange with the engineer trying to
> solve the
> > problem in remote,
> > it turns out that this is something more serious. The engineer comes
> over, collects
> > the laser and promise we will have the laser back in 1,5-2 months. End
> of May
> > then. That is the only Multi Photon laser in the facility and I start
> already to have
> > worried users around asking when the laser will be back.
> >
> > 03.06.2011 I write Spectra Physics (SP) urging news about the laser.
> >
> > 27.06.2011 SP informs me that they will start to work on the laser "this
> week" and
> > the laser should be back in the week of the 11th of July. They added "If
> it is
> > possible to do it any earlier we will".
> >
> > 01.07.2011 SP informs me that there are good news: the laser will be
> really back by
> > the middle of July.
> >
> > 14.07.2011 No news from SP. I write them asking about an update. SP
> answers
> > quickly: new installation date, the 3rd of August. 2011? My bad, I forgot
> to ask
> > them.
> >
> > 05.08.2011 Again no news and no laser back. I write this time the Sales
> Director a
> > complaint letter asking when we will have our laser back and if it is
> possible to
> > have a replacement instrument, as we are without laser since more than 4
> months
> > now.
> >
> > 17.08.2011 Receiving no answer from the Sales Director I call him, and
> receive
> > assurance that the laser will be back next week.
> >
> > 26.08.2011 Still nothing. I write a new complaint mail to the Sales
> Director. He
> > answers that there were more delays, but by the 31st of August he will
> surely be
> > able to know more about when the laser will be back and he will contact
> me.
> >
> > 01.09.2011 No news from SP (to be continued).
> >
> > Now it might well be that all of it is absolutely normal, I just would
> like to get a
> > feedback from the community if there is anything else I can do in order
> to have the
> > laser back.
> > Thanks for reading this overly long post and for your feedback.
> >
> > Cheers,
> >
> > Marco
> >
> >
> > Marco Marcello
> > Imaging Manager
> > Centre for Cellular Imaging, IIB
> > University of Liverpool
> > UK
>

Armstrong, Brian

Re: Newport MaiTai 2-photon laser maintenance

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I agree that service is region specific and that you should check with people in your region before purchasing an instrument. Let me share my experience with Coherent service.

Our Coherent service rep came by when I reported that I thought the power had been dropping on one of our Chameleon lasers. It still had plenty of power, but I thought it may be a symptom of a bigger problem such as a inefficient chiller. The service rep thought that the Verdi power was low so he decided to replace the entire laser system. A new refurbished Chameleon laser came in a large wooden crate about 3 weeks later. We placed the old Chameleon in the wooden crate for return shipment and installed the new Chameleon laser system. We were without the use of the laser for about 1 hour. We have had excellent service from Coherent, but then again we maintain a service contract which may greatly influence the service responsiveness.

Cheers,

Brian Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
1500 East Duarte Road
Duarte, CA 91010
626-256-4673 x62872

Light Microscopy Core Facility

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Thursday, September 01, 2011 9:52 AM
To: [hidden email]
Subject: Re: Newport MaiTai 2-photon laser maintenance

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Service from any company seems to be very hit-and-miss these days. I have
noticed severe slowdowns whenever I need anything done from almost every
company. From what I have heard, support tends to be one of the first
things that gets cut when a company needs to downsize. As a result, all the
existing customers suffer. I keep hearing stories about how this or that
service rep or even manager has been laid off! The key thing is to find out
how service is in your local area with regards to whatever company you are
considering purchasing from. If you are unlucky, the service rep in your
area will either have been laid off, or is so badly overworked he'll never
have the time to get to you.'

Craig

On Thu, Sep 1, 2011 at 5:34 AM, Sylvie LeGuyader <[hidden email]>wrote:

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mcammer

Re: Newport MaiTai 2-photon laser maintenance

In reply to this post by Craig Brideau

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First I'd like to say that we have excellent service support from Spectraphysics, so this is not about them in any way.

But as a general policy, any company that does not successfully address service issues for equipment under warrantee or under a service contract within the terms of the agreement or in a reasonable time should be sued. The company should be expected to remedy the situation, pay damages, and cover all attorney's fees. Your institution either has in-house counsel or a retainer with attorneys standing by waiting for your call.

-Michael

On Thu, Sep 1, 2011 at 5:34 AM, Sylvie LeGuyader <[hidden email]>wrote:

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