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recommended distortion target for multiphoton?

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Tobias Rose

Re: recommended distortion target for multiphoton?

My thorlabs slide is also a total mess by now. Even though I could use reflected light I am usually too lazy to get rid of my nearIR filters.

One thing that I've used before for 2p field size calibration (and will use again once I evaporated the last bits of visible grid on my thorlabs slide) are Neubauer chambers (http://en.wikipedia.org/wiki/Hemocytometer) used for counting blood cells. These are rather cheap and nearly indestructible.

Neubauer chambers have calibrated grid channels deeply etched into the thick glass. One can carefully break the chamber into small pieces and submerge these in fluorescent solution. When focused on the channel,s one gets a very nice 2p fluorescent grid.

Cheers,
Tobias

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Michael
> Giacomelli
> Sent: Tuesday, September 23, 2014 00:49
> To: [hidden email]
> Subject: Re: recommended distortion target for multiphoton?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Just to follow up, I contacted MicronLaser.com and got several 1 cm^2 grid
> target etched into borosilicate glass. They are ~20 um deep and so I can flow
> different fluorophores through. The cost was very reasonable, but they were
> machined with a CO2 laser, and so have some thermal microcracking that
> makes them unsuitable as resolution targets. Still, they are virtually
> indestructable (can park the beam forever at one point and you'll never damage
> it), and the very large size makes them quite handy for testing translation
> stages and mosaicing software.
>
> I'd still like to find some who can do a nanosecond pulsed laser etch and see
> about getting custom resolution targets cut, but overall, the micronlaser parts
> were quite reasonable for what I needed.
>
> Mike
>
> On Wed, Jul 23, 2014 at 2:00 PM, Craig Brideau <[hidden email]>
> wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your posting.
> > *****
> >
> > I've been bugging various companies for years to get a good 2P and
> > confocal fluoresence target developed, but thus far nobody's come up
> > with anything that really works that costs less than $1000.
> >
> > Craig Brideau
> >
> >
> > On Wed, Jul 23, 2014 at 11:26 AM, Michael Giacomelli <[hidden email]>
> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your posting.
> >> *****
> >>
> >> Unfortunately, it is extremely difficult to record the reflection
> >> mode image in our microscope, as the NIR light will not pass through
> >> the dichroic, and even if we changed out the dichroic for a beam
> >> splitter, out PMTs are not sensitive to NIR light.
> >>
> >> Translating a bead though is a very interesting idea. We use a high
> >> precision thorlabs MLS stage. I will double check the repeatability,
> >> but in theory I could program it to perform an extremely high
> >> resolution distortion measurement. Of course, getting a single bead
> >> onto a coverslip (or else programming software to automatically
> >> identify the same bead across hundreds of frames) will be somewhat
> >> tricky.
> >>
> >> Its a shame that getting targets for multiphoton is so difficult.
> >> Ideal I suppose would be something etched in glass so that I could
> >> flow my own fluorophore through it. I will have to look around and
> >> see if I can get a lithography facility to make something like that.
> >>
> >> Thanks,
> >> Mike
> >>
> >> On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
> >> <[hidden email]> wrote:
> >> > *****
> >> > To join, leave or search the confocal microscopy listserv, go to:
> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> > Post images on http://www.imgur.com and include the link in your
> >> posting.
> >> > *****
> >> >
> >> > Thinking about this a bit more, I realised that your existing grid
> >> > is
> >> perfectly suitable as you don’t need 2P excitation to measure field
> >> distortion at the wavelength you are using, Just turn the power down
> >> a lot and record a reflection image,
> >> >
> >> > Cheers Mark
> >> >
> >> >>>
> >> >>> Hi Mark,
> >> >>>
> >> >>> I'm assuming you mean beads deposited in a grid or something similar?
> >> >>> Could you point me to where you found this?
> >> >>>
> >> >>> Thanks,
> >> >>> Mike
> >> >>>
> >> >>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
> >> >>> <[hidden email]> wrote:
> >> >>>> *****
> >> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >>>> Post images on http://www.imgur.com and include the link in your
> >> >>> posting.
> >> >>>> *****
> >> >>>>
> >> >>>> Hi Michael
> >> >>>>
> >> >>>> I’ve always used beads to test the microscope. They are
> >> >>>> relatively
> >> cheap
> >> >>> and if you blow one up it don’t matter. As you say, evaporated
> >> >>> metal targets are too easily damaged (as Jim Pauley and I
> >> >>> discovered when
> >> testing
> >> >>> my microscope back in ’95!)
> >> >>>>
> >> >>>> HTH
> >> >>>>
> >> >>>> Mark
> >> >>>>
> >> >>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]>
> wrote:
> >> >>>>
> >> >>>>> *****
> >> >>>>> To join, leave or search the confocal microscopy listserv, go to:
> >> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >>>>> Post images on http://www.imgur.com and include the link in
> >> >>>>> your
> >> >>> posting.
> >> >>>>> *****
> >> >>>>>
> >> >>>>> Could someone recommend a good multiphoton distortion target?
> >> >>>>> Preferably one that is relatively robust against damage.
> >> >>>>>
> >> >>>>> Currently I am using a thorlabs wire grid target with
> >> >>>>> fluorophore behind it. The beam is attenuated by the metal,
> >> >>>>> resulting in an
> >> image
> >> >>>>> of the grid. However, passing through the entire 1 mm slide
> >> >>>>> greatly reduces my resolution, and unless I am extremely
> >> >>>>> careful, the grid is ablated by the beam.
> >> >>>>>
> >> >>>>> Thanks,
> >> >>>>> Michael
> >> >>>>
> >> >>>>
> >> >>>> Mark B. Cannell Ph.D. FRSNZ
> >> >>>> Professor of Cardiac Cell Biology School of Physiology &
> >> >>>> Pharmacology Medical Sciences Building University of Bristol
> >> >>>> Bristol
> >> >>>> BS8 1TD UK
> >> >>>>
> >> >>>> [hidden email]
> >> >>>
> >> >
> >> >
> >> >
> >> > Mark B. Cannell Ph.D. FRSNZ
> >> > Professor of Cardiac Cell Biology
> >> > School of Physiology & Pharmacology Medical Sciences Building
> >> > University of Bristol Bristol
> >> > BS8 1TD UK
> >> >
> >> > [hidden email]
> >>

James Pawley

Re: recommended distortion target for multiphoton?

In reply to this post by Michael Giacomelli

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Just to follow on with what Mark said, if all you want is to measure
distortion, you don't even need a reflection image of your grid. Just
use a transmission detector and, if you don't have one (assuming
inverted optics), a PIN diode sitting on the top of the slide should
do nicely, as TPE usually has a massive (mW) beam.

JP

>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Unfortunately, it is extremely difficult to record the reflection mode
>image in our microscope, as the NIR light will not pass through the
>dichroic, and even if we changed out the dichroic for a beam splitter,
>out PMTs are not sensitive to NIR light.
>
>Translating a bead though is a very interesting idea. We use a high
>precision thorlabs MLS stage. I will double check the repeatability,
>but in theory I could program it to perform an extremely high
>resolution distortion measurement. Of course, getting a single bead
>onto a coverslip (or else programming software to automatically
>identify the same bead across hundreds of frames) will be somewhat
>tricky.
>
>Its a shame that getting targets for multiphoton is so difficult.
>Ideal I suppose would be something etched in glass so that I could
>flow my own fluorophore through it. I will have to look around and
>see if I can get a lithography facility to make something like that.
>
>Thanks,
>Mike
>
>On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
><[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Thinking about this a bit more, I realised that your existing grid
>>is perfectly suitable as you don't need 2P excitation to measure
>>field distortion at the wavelength you are using, Just turn the
>>power down a lot and record a reflection image,
>>
>> Cheers Mark
>>
>>>>
>>>> Hi Mark,
>>>>
>>>> I'm assuming you mean beads deposited in a grid or something similar?
>>>> Could you point me to where you found this?
>>>>
>>>> Thanks,
>>>> Mike
>>>>
>>>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
>>>> <[hidden email]> wrote:
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>>> *****
>>>>>
>>>>> Hi Michael
>>>>>
>>>>> I've always used beads to test the microscope. They are relatively cheap
>>>> and if you blow one up it don't matter. As you say, evaporated metal
>>>> targets are too easily damaged (as Jim Pauley and I discovered
>>>>when testing
>>>> my microscope back in '95!)
>>>>>
>>>>> HTH
>>>>>
>>>>> Mark
>>>>>
>>>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]> wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>>>> *****
>>>>>>
>>>>>> Could someone recommend a good multiphoton distortion target?
>>>>>> Preferably one that is relatively robust against damage.
>>>>>>
>>>>>> Currently I am using a thorlabs wire grid target with fluorophore
>>>>>> behind it. The beam is attenuated by the metal, resulting in an image
>>>>>> of the grid. However, passing through the entire 1 mm slide greatly
>>>>>> reduces my resolution, and unless I am extremely careful, the grid is
>>>>>> ablated by the beam.
>>>>>>
>>>>>> Thanks,
>>>>>> Michael
>>>>>
>>>>>
>>>>> Mark B. Cannell Ph.D. FRSNZ
>>>>> Professor of Cardiac Cell Biology
>>>>> School of Physiology & Pharmacology
>>>>> Medical Sciences Building
>>>>> University of Bristol
>>>>> Bristol
>>>>> BS8 1TD UK
>>>>>
>>>>> [hidden email]
> >>>
>>
>>
>>
>> Mark B. Cannell Ph.D. FRSNZ
>> Professor of Cardiac Cell Biology
>> School of Physiology & Pharmacology
>> Medical Sciences Building
>> University of Bristol
>> Bristol
>> BS8 1TD UK
>>
>> [hidden email]

--
Prof. James Pawley, Shangri La Hotel, Harbin,
email <[hidden email]>, China Cell phone 15636810175

Michael Giacomelli

Re: recommended distortion target for multiphoton?

In reply to this post by Tobias Rose

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Tobias,

Could you recommend a hemocytometer to use? I'd tried one of the ones
we had in lab months ago, but the etching in it was only a few hundred
nanometers deep, making it extremely hard to see on MP. I just
assumed all were like that, but apparently not.

Mike

On Tue, Sep 23, 2014 at 2:31 AM, Tobias Rose <[hidden email]> wrote:

> My thorlabs slide is also a total mess by now. Even though I could use reflected light I am usually too lazy to get rid of my nearIR filters.
>
> One thing that I've used before for 2p field size calibration (and will use again once I evaporated the last bits of visible grid on my thorlabs slide) are Neubauer chambers (http://en.wikipedia.org/wiki/Hemocytometer) used for counting blood cells. These are rather cheap and nearly indestructible.
>
> Neubauer chambers have calibrated grid channels deeply etched into the thick glass. One can carefully break the chamber into small pieces and submerge these in fluorescent solution. When focused on the channel,s one gets a very nice 2p fluorescent grid.
>
> Cheers,
> Tobias
>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Michael
>> Giacomelli
>> Sent: Tuesday, September 23, 2014 00:49
>> To: [hidden email]
>> Subject: Re: recommended distortion target for multiphoton?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Just to follow up, I contacted MicronLaser.com and got several 1 cm^2 grid
>> target etched into borosilicate glass. They are ~20 um deep and so I can flow
>> different fluorophores through. The cost was very reasonable, but they were
>> machined with a CO2 laser, and so have some thermal microcracking that
>> makes them unsuitable as resolution targets. Still, they are virtually
>> indestructable (can park the beam forever at one point and you'll never damage
>> it), and the very large size makes them quite handy for testing translation
>> stages and mosaicing software.
>>
>> I'd still like to find some who can do a nanosecond pulsed laser etch and see
>> about getting custom resolution targets cut, but overall, the micronlaser parts
>> were quite reasonable for what I needed.
>>
>> Mike
>>
>> On Wed, Jul 23, 2014 at 2:00 PM, Craig Brideau <[hidden email]>
>> wrote:
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > Post images on http://www.imgur.com and include the link in your posting.
>> > *****
>> >
>> > I've been bugging various companies for years to get a good 2P and
>> > confocal fluoresence target developed, but thus far nobody's come up
>> > with anything that really works that costs less than $1000.
>> >
>> > Craig Brideau
>> >
>> >
>> > On Wed, Jul 23, 2014 at 11:26 AM, Michael Giacomelli <[hidden email]>
>> wrote:
>> >
>> >> *****
>> >> To join, leave or search the confocal microscopy listserv, go to:
>> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >> Post images on http://www.imgur.com and include the link in your posting.
>> >> *****
>> >>
>> >> Unfortunately, it is extremely difficult to record the reflection
>> >> mode image in our microscope, as the NIR light will not pass through
>> >> the dichroic, and even if we changed out the dichroic for a beam
>> >> splitter, out PMTs are not sensitive to NIR light.
>> >>
>> >> Translating a bead though is a very interesting idea. We use a high
>> >> precision thorlabs MLS stage. I will double check the repeatability,
>> >> but in theory I could program it to perform an extremely high
>> >> resolution distortion measurement. Of course, getting a single bead
>> >> onto a coverslip (or else programming software to automatically
>> >> identify the same bead across hundreds of frames) will be somewhat
>> >> tricky.
>> >>
>> >> Its a shame that getting targets for multiphoton is so difficult.
>> >> Ideal I suppose would be something etched in glass so that I could
>> >> flow my own fluorophore through it. I will have to look around and
>> >> see if I can get a lithography facility to make something like that.
>> >>
>> >> Thanks,
>> >> Mike
>> >>
>> >> On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
>> >> <[hidden email]> wrote:
>> >> > *****
>> >> > To join, leave or search the confocal microscopy listserv, go to:
>> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >> > Post images on http://www.imgur.com and include the link in your
>> >> posting.
>> >> > *****
>> >> >
>> >> > Thinking about this a bit more, I realised that your existing grid
>> >> > is
>> >> perfectly suitable as you don’t need 2P excitation to measure field
>> >> distortion at the wavelength you are using, Just turn the power down
>> >> a lot and record a reflection image,
>> >> >
>> >> > Cheers Mark
>> >> >
>> >> >>>
>> >> >>> Hi Mark,
>> >> >>>
>> >> >>> I'm assuming you mean beads deposited in a grid or something similar?
>> >> >>> Could you point me to where you found this?
>> >> >>>
>> >> >>> Thanks,
>> >> >>> Mike
>> >> >>>
>> >> >>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
>> >> >>> <[hidden email]> wrote:
>> >> >>>> *****
>> >> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >> >>>> Post images on http://www.imgur.com and include the link in your
>> >> >>> posting.
>> >> >>>> *****
>> >> >>>>
>> >> >>>> Hi Michael
>> >> >>>>
>> >> >>>> I’ve always used beads to test the microscope. They are
>> >> >>>> relatively
>> >> cheap
>> >> >>> and if you blow one up it don’t matter. As you say, evaporated
>> >> >>> metal targets are too easily damaged (as Jim Pauley and I
>> >> >>> discovered when
>> >> testing
>> >> >>> my microscope back in ’95!)
>> >> >>>>
>> >> >>>> HTH
>> >> >>>>
>> >> >>>> Mark
>> >> >>>>
>> >> >>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]>
>> wrote:
>> >> >>>>
>> >> >>>>> *****
>> >> >>>>> To join, leave or search the confocal microscopy listserv, go to:
>> >> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >> >>>>> Post images on http://www.imgur.com and include the link in
>> >> >>>>> your
>> >> >>> posting.
>> >> >>>>> *****
>> >> >>>>>
>> >> >>>>> Could someone recommend a good multiphoton distortion target?
>> >> >>>>> Preferably one that is relatively robust against damage.
>> >> >>>>>
>> >> >>>>> Currently I am using a thorlabs wire grid target with
>> >> >>>>> fluorophore behind it. The beam is attenuated by the metal,
>> >> >>>>> resulting in an
>> >> image
>> >> >>>>> of the grid. However, passing through the entire 1 mm slide
>> >> >>>>> greatly reduces my resolution, and unless I am extremely
>> >> >>>>> careful, the grid is ablated by the beam.
>> >> >>>>>
>> >> >>>>> Thanks,
>> >> >>>>> Michael
>> >> >>>>
>> >> >>>>
>> >> >>>> Mark B. Cannell Ph.D. FRSNZ
>> >> >>>> Professor of Cardiac Cell Biology School of Physiology &
>> >> >>>> Pharmacology Medical Sciences Building University of Bristol
>> >> >>>> Bristol
>> >> >>>> BS8 1TD UK
>> >> >>>>
>> >> >>>> [hidden email]
>> >> >>>
>> >> >
>> >> >
>> >> >
>> >> > Mark B. Cannell Ph.D. FRSNZ
>> >> > Professor of Cardiac Cell Biology
>> >> > School of Physiology & Pharmacology Medical Sciences Building
>> >> > University of Bristol Bristol
>> >> > BS8 1TD UK
>> >> >
>> >> > [hidden email]
>> >>

Tobias Rose

Re: recommended distortion target for multiphoton?

Hi Mike,

Good question. I, in turn, assumed all were like the ones I used back in my old lab... I will check and come back to you.

Tobias

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Michael
> Giacomelli
> Sent: Sunday, October 05, 2014 02:36
> To: [hidden email]
> Subject: Re: recommended distortion target for multiphoton?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Tobias,
>
> Could you recommend a hemocytometer to use? I'd tried one of the ones we
> had in lab months ago, but the etching in it was only a few hundred nanometers
> deep, making it extremely hard to see on MP. I just assumed all were like that,
> but apparently not.
>
> Mike
>
> On Tue, Sep 23, 2014 at 2:31 AM, Tobias Rose <[hidden email]> wrote:
> > My thorlabs slide is also a total mess by now. Even though I could use
> reflected light I am usually too lazy to get rid of my nearIR filters.
> >
> > One thing that I've used before for 2p field size calibration (and will use again
> once I evaporated the last bits of visible grid on my thorlabs slide) are
> Neubauer chambers (http://en.wikipedia.org/wiki/Hemocytometer) used for
> counting blood cells. These are rather cheap and nearly indestructible.
> >
> > Neubauer chambers have calibrated grid channels deeply etched into the
> thick glass. One can carefully break the chamber into small pieces and
> submerge these in fluorescent solution. When focused on the channel,s one
> gets a very nice 2p fluorescent grid.
> >
> > Cheers,
> > Tobias
> >
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[hidden email]] On Behalf Of Michael
> >> Giacomelli
> >> Sent: Tuesday, September 23, 2014 00:49
> >> To: [hidden email]
> >> Subject: Re: recommended distortion target for multiphoton?
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your posting.
> >> *****
> >>
> >> Just to follow up, I contacted MicronLaser.com and got several 1 cm^2
> >> grid target etched into borosilicate glass. They are ~20 um deep and
> >> so I can flow different fluorophores through. The cost was very
> >> reasonable, but they were machined with a CO2 laser, and so have some
> >> thermal microcracking that makes them unsuitable as resolution
> >> targets. Still, they are virtually indestructable (can park the beam
> >> forever at one point and you'll never damage it), and the very large
> >> size makes them quite handy for testing translation stages and mosaicing
> software.
> >>
> >> I'd still like to find some who can do a nanosecond pulsed laser etch
> >> and see about getting custom resolution targets cut, but overall, the
> >> micronlaser parts were quite reasonable for what I needed.
> >>
> >> Mike
> >>
> >> On Wed, Jul 23, 2014 at 2:00 PM, Craig Brideau
> >> <[hidden email]>
> >> wrote:
> >> > *****
> >> > To join, leave or search the confocal microscopy listserv, go to:
> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> > Post images on http://www.imgur.com and include the link in your posting.
> >> > *****
> >> >
> >> > I've been bugging various companies for years to get a good 2P and
> >> > confocal fluoresence target developed, but thus far nobody's come
> >> > up with anything that really works that costs less than $1000.
> >> >
> >> > Craig Brideau
> >> >
> >> >
> >> > On Wed, Jul 23, 2014 at 11:26 AM, Michael Giacomelli <[hidden email]>
> >> wrote:
> >> >
> >> >> *****
> >> >> To join, leave or search the confocal microscopy listserv, go to:
> >> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> >> *****
> >> >>
> >> >> Unfortunately, it is extremely difficult to record the reflection
> >> >> mode image in our microscope, as the NIR light will not pass
> >> >> through the dichroic, and even if we changed out the dichroic for
> >> >> a beam splitter, out PMTs are not sensitive to NIR light.
> >> >>
> >> >> Translating a bead though is a very interesting idea. We use a
> >> >> high precision thorlabs MLS stage. I will double check the
> >> >> repeatability, but in theory I could program it to perform an
> >> >> extremely high resolution distortion measurement. Of course,
> >> >> getting a single bead onto a coverslip (or else programming
> >> >> software to automatically identify the same bead across hundreds
> >> >> of frames) will be somewhat tricky.
> >> >>
> >> >> Its a shame that getting targets for multiphoton is so difficult.
> >> >> Ideal I suppose would be something etched in glass so that I could
> >> >> flow my own fluorophore through it. I will have to look around
> >> >> and see if I can get a lithography facility to make something like that.
> >> >>
> >> >> Thanks,
> >> >> Mike
> >> >>
> >> >> On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
> >> >> <[hidden email]> wrote:
> >> >> > *****
> >> >> > To join, leave or search the confocal microscopy listserv, go to:
> >> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >> > Post images on http://www.imgur.com and include the link in your
> >> >> posting.
> >> >> > *****
> >> >> >
> >> >> > Thinking about this a bit more, I realised that your existing
> >> >> > grid is
> >> >> perfectly suitable as you don’t need 2P excitation to measure
> >> >> field distortion at the wavelength you are using, Just turn the
> >> >> power down a lot and record a reflection image,
> >> >> >
> >> >> > Cheers Mark
> >> >> >
> >> >> >>>
> >> >> >>> Hi Mark,
> >> >> >>>
> >> >> >>> I'm assuming you mean beads deposited in a grid or something
> similar?
> >> >> >>> Could you point me to where you found this?
> >> >> >>>
> >> >> >>> Thanks,
> >> >> >>> Mike
> >> >> >>>
> >> >> >>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
> >> >> >>> <[hidden email]> wrote:
> >> >> >>>> *****
> >> >> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >> >> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >> >>>> Post images on http://www.imgur.com and include the link in
> >> >> >>>> your
> >> >> >>> posting.
> >> >> >>>> *****
> >> >> >>>>
> >> >> >>>> Hi Michael
> >> >> >>>>
> >> >> >>>> I’ve always used beads to test the microscope. They are
> >> >> >>>> relatively
> >> >> cheap
> >> >> >>> and if you blow one up it don’t matter. As you say, evaporated
> >> >> >>> metal targets are too easily damaged (as Jim Pauley and I
> >> >> >>> discovered when
> >> >> testing
> >> >> >>> my microscope back in ’95!)
> >> >> >>>>
> >> >> >>>> HTH
> >> >> >>>>
> >> >> >>>> Mark
> >> >> >>>>
> >> >> >>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]>
> >> wrote:
> >> >> >>>>
> >> >> >>>>> *****
> >> >> >>>>> To join, leave or search the confocal microscopy listserv, go to:
> >> >> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> >> >>>>> Post images on http://www.imgur.com and include the link in
> >> >> >>>>> your
> >> >> >>> posting.
> >> >> >>>>> *****
> >> >> >>>>>
> >> >> >>>>> Could someone recommend a good multiphoton distortion target?
> >> >> >>>>> Preferably one that is relatively robust against damage.
> >> >> >>>>>
> >> >> >>>>> Currently I am using a thorlabs wire grid target with
> >> >> >>>>> fluorophore behind it. The beam is attenuated by the metal,
> >> >> >>>>> resulting in an
> >> >> image
> >> >> >>>>> of the grid. However, passing through the entire 1 mm slide
> >> >> >>>>> greatly reduces my resolution, and unless I am extremely
> >> >> >>>>> careful, the grid is ablated by the beam.
> >> >> >>>>>
> >> >> >>>>> Thanks,
> >> >> >>>>> Michael
> >> >> >>>>
> >> >> >>>>
> >> >> >>>> Mark B. Cannell Ph.D. FRSNZ
> >> >> >>>> Professor of Cardiac Cell Biology School of Physiology &
> >> >> >>>> Pharmacology Medical Sciences Building University of Bristol
> >> >> >>>> Bristol
> >> >> >>>> BS8 1TD UK
> >> >> >>>>
> >> >> >>>> [hidden email]
> >> >> >>>
> >> >> >
> >> >> >
> >> >> >
> >> >> > Mark B. Cannell Ph.D. FRSNZ
> >> >> > Professor of Cardiac Cell Biology School of Physiology &
> >> >> > Pharmacology Medical Sciences Building University of Bristol
> >> >> > Bristol
> >> >> > BS8 1TD UK
> >> >> >
> >> >> > [hidden email]
> >> >>

Julio MATEOS_LANGERAK

Re: recommended distortion target for multiphoton?

In reply to this post by Michael Giacomelli

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi,

You might have a look at Argolight target slides. I don't know how they perform with multiphoton systems though.

Best, Julio

Julio Mateos Langerak, PhD.
OMX manager
Arnaud de Villeneuve Campus Imaging Facility
Montpellier RIO Imaging
Montpellier BIOCAMPUS, UMS3426
Institut de Génétique Humaine-CNRS
141, rue de la Cardonille
F-34396 Montpellier (France)
e-mail: [hidden email]: +33.4.34.35.99.90
fax: +33.4.34.35.99.01

Sent from my iPhone

> On 5 Oct 2014, at 02:36, Michael Giacomelli <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Tobias,
>
> Could you recommend a hemocytometer to use? I'd tried one of the ones
> we had in lab months ago, but the etching in it was only a few hundred
> nanometers deep, making it extremely hard to see on MP. I just
> assumed all were like that, but apparently not.
>
> Mike
>
>> On Tue, Sep 23, 2014 at 2:31 AM, Tobias Rose <[hidden email]> wrote:
>> My thorlabs slide is also a total mess by now. Even though I could use reflected light I am usually too lazy to get rid of my nearIR filters.
>>
>> One thing that I've used before for 2p field size calibration (and will use again once I evaporated the last bits of visible grid on my thorlabs slide) are Neubauer chambers (http://en.wikipedia.org/wiki/Hemocytometer) used for counting blood cells. These are rather cheap and nearly indestructible.
>>
>> Neubauer chambers have calibrated grid channels deeply etched into the thick glass. One can carefully break the chamber into small pieces and submerge these in fluorescent solution. When focused on the channel,s one gets a very nice 2p fluorescent grid.
>>
>> Cheers,
>> Tobias
>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]] On Behalf Of Michael
>>> Giacomelli
>>> Sent: Tuesday, September 23, 2014 00:49
>>> To: [hidden email]
>>> Subject: Re: recommended distortion target for multiphoton?
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> Just to follow up, I contacted MicronLaser.com and got several 1 cm^2 grid
>>> target etched into borosilicate glass. They are ~20 um deep and so I can flow
>>> different fluorophores through. The cost was very reasonable, but they were
>>> machined with a CO2 laser, and so have some thermal microcracking that
>>> makes them unsuitable as resolution targets. Still, they are virtually
>>> indestructable (can park the beam forever at one point and you'll never damage
>>> it), and the very large size makes them quite handy for testing translation
>>> stages and mosaicing software.
>>>
>>> I'd still like to find some who can do a nanosecond pulsed laser etch and see
>>> about getting custom resolution targets cut, but overall, the micronlaser parts
>>> were quite reasonable for what I needed.
>>>
>>> Mike
>>>
>>> On Wed, Jul 23, 2014 at 2:00 PM, Craig Brideau <[hidden email]>
>>> wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your posting.
>>>> *****
>>>>
>>>> I've been bugging various companies for years to get a good 2P and
>>>> confocal fluoresence target developed, but thus far nobody's come up
>>>> with anything that really works that costs less than $1000.
>>>>
>>>> Craig Brideau
>>>>
>>>>
>>>>> On Wed, Jul 23, 2014 at 11:26 AM, Michael Giacomelli <[hidden email]>
>>>> wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your posting.
>>>>> *****
>>>>>
>>>>> Unfortunately, it is extremely difficult to record the reflection
>>>>> mode image in our microscope, as the NIR light will not pass through
>>>>> the dichroic, and even if we changed out the dichroic for a beam
>>>>> splitter, out PMTs are not sensitive to NIR light.
>>>>>
>>>>> Translating a bead though is a very interesting idea. We use a high
>>>>> precision thorlabs MLS stage. I will double check the repeatability,
>>>>> but in theory I could program it to perform an extremely high
>>>>> resolution distortion measurement. Of course, getting a single bead
>>>>> onto a coverslip (or else programming software to automatically
>>>>> identify the same bead across hundreds of frames) will be somewhat
>>>>> tricky.
>>>>>
>>>>> Its a shame that getting targets for multiphoton is so difficult.
>>>>> Ideal I suppose would be something etched in glass so that I could
>>>>> flow my own fluorophore through it. I will have to look around and
>>>>> see if I can get a lithography facility to make something like that.
>>>>>
>>>>> Thanks,
>>>>> Mike
>>>>>
>>>>> On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
>>>>> <[hidden email]> wrote:
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>>> *****
>>>>>>
>>>>>> Thinking about this a bit more, I realised that your existing grid
>>>>>> is
>>>>> perfectly suitable as you don’t need 2P excitation to measure field
>>>>> distortion at the wavelength you are using, Just turn the power down
>>>>> a lot and record a reflection image,
>>>>>>
>>>>>> Cheers Mark
>>>>>>
>>>>>>>>
>>>>>>>> Hi Mark,
>>>>>>>>
>>>>>>>> I'm assuming you mean beads deposited in a grid or something similar?
>>>>>>>> Could you point me to where you found this?
>>>>>>>>
>>>>>>>> Thanks,
>>>>>>>> Mike
>>>>>>>>
>>>>>>>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
>>>>>>>> <[hidden email]> wrote:
>>>>>>>>> *****
>>>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>>>> posting.
>>>>>>>>> *****
>>>>>>>>>
>>>>>>>>> Hi Michael
>>>>>>>>>
>>>>>>>>> I’ve always used beads to test the microscope. They are
>>>>>>>>> relatively
>>>>> cheap
>>>>>>>> and if you blow one up it don’t matter. As you say, evaporated
>>>>>>>> metal targets are too easily damaged (as Jim Pauley and I
>>>>>>>> discovered when
>>>>> testing
>>>>>>>> my microscope back in ’95!)
>>>>>>>>>
>>>>>>>>> HTH
>>>>>>>>>
>>>>>>>>> Mark
>>>>>>>>>
>>>>>>>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]>
>>> wrote:
>>>>>>>>>
>>>>>>>>>> *****
>>>>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>>>> Post images on http://www.imgur.com and include the link in
>>>>>>>>>> your
>>>>>>>> posting.
>>>>>>>>>> *****
>>>>>>>>>>
>>>>>>>>>> Could someone recommend a good multiphoton distortion target?
>>>>>>>>>> Preferably one that is relatively robust against damage.
>>>>>>>>>>
>>>>>>>>>> Currently I am using a thorlabs wire grid target with
>>>>>>>>>> fluorophore behind it. The beam is attenuated by the metal,
>>>>>>>>>> resulting in an
>>>>> image
>>>>>>>>>> of the grid. However, passing through the entire 1 mm slide
>>>>>>>>>> greatly reduces my resolution, and unless I am extremely
>>>>>>>>>> careful, the grid is ablated by the beam.
>>>>>>>>>>
>>>>>>>>>> Thanks,
>>>>>>>>>> Michael
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> Mark B. Cannell Ph.D. FRSNZ
>>>>>>>>> Professor of Cardiac Cell Biology School of Physiology &
>>>>>>>>> Pharmacology Medical Sciences Building University of Bristol
>>>>>>>>> Bristol
>>>>>>>>> BS8 1TD UK
>>>>>>>>>
>>>>>>>>> [hidden email]
>>>>>>
>>>>>>
>>>>>>
>>>>>> Mark B. Cannell Ph.D. FRSNZ
>>>>>> Professor of Cardiac Cell Biology
>>>>>> School of Physiology & Pharmacology Medical Sciences Building
>>>>>> University of Bristol Bristol
>>>>>> BS8 1TD UK
>>>>>>
>>>>>> [hidden email]
>>>>>

Tobias Rose

Re: recommended distortion target for multiphoton?

In reply to this post by Michael Giacomelli

Hi Mike,

This is embarrassing - I need to take back my Hemocytometer suggestion...

We either used Neubauer chambers from a Swiss company called "Brand" or from the German company E. Hartnack (Berlin) (Thx to Simon Wiegert: http://www.evernote.com/l/AATsa1AjNZpPk5Lna0rWUSglekMYLdCYesc/).

BUT:
It turns out that my memory played a trick on me. We generally used them in transmitted light mode (diode detection of scanned IR laser through the condenser).
I therefore cannot guarantee that the etched grid lines are deep enough for fluorescence measurements. I'm still rather convinced that I used them also like this - but this may be my brain post-hoc rationalizing things. When I get my hand on a chamber again, I will check.

Sorry for being misleading.

T

Michael Giacomelli

Re: recommended distortion target for multiphoton?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Tobias,

Looking around Google, I see a few references to using them with confocals
for calibration, so presumably some are deep enough etched to be visible.
Unfortunately, short of buying/borrowing and testing, I'm not sure how to
find a suitable one.

Mike

On Thu, Oct 9, 2014 at 6:17 AM, Tobias Rose <[hidden email]> wrote:

> Hi Mike,
>
> This is embarrassing - I need to take back my Hemocytometer suggestion...
>
> We either used Neubauer chambers from a Swiss company called "Brand" or
> from the German company E. Hartnack (Berlin) (Thx to Simon Wiegert:
> http://www.evernote.com/l/AATsa1AjNZpPk5Lna0rWUSglekMYLdCYesc/).
>
> BUT:
> It turns out that my memory played a trick on me. We generally used them
> in transmitted light mode (diode detection of scanned IR laser through the
> condenser).
> I therefore cannot guarantee that the etched grid lines are deep enough
> for fluorescence measurements. I'm still rather convinced that I used them
> also like this - but this may be my brain post-hoc rationalizing things.
> When I get my hand on a chamber again, I will check.
>
> Sorry for being misleading.
>
> T
>
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]] On Behalf Of Michael
> > Giacomelli
> > Sent: Sunday, October 05, 2014 02:36
> > To: [hidden email]
> > Subject: Re: recommended distortion target for multiphoton?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Tobias,
> >
> > Could you recommend a hemocytometer to use? I'd tried one of the ones we
> > had in lab months ago, but the etching in it was only a few hundred
> nanometers
> > deep, making it extremely hard to see on MP. I just assumed all were
> like that,
> > but apparently not.
> >
> > Mike
> >
> > On Tue, Sep 23, 2014 at 2:31 AM, Tobias Rose <[hidden email]> wrote:
> > > My thorlabs slide is also a total mess by now. Even though I could use
> > reflected light I am usually too lazy to get rid of my nearIR filters.
> > >
> > > One thing that I've used before for 2p field size calibration (and
> will use again
> > once I evaporated the last bits of visible grid on my thorlabs slide) are
> > Neubauer chambers (http://en.wikipedia.org/wiki/Hemocytometer) used for
> > counting blood cells. These are rather cheap and nearly indestructible.
> > >
> > > Neubauer chambers have calibrated grid channels deeply etched into the
> > thick glass. One can carefully break the chamber into small pieces and
> > submerge these in fluorescent solution. When focused on the channel,s one
> > gets a very nice 2p fluorescent grid.
> > >
> > > Cheers,
> > > Tobias
> > >
> > >> -----Original Message-----
> > >> From: Confocal Microscopy List
> > >> [mailto:[hidden email]] On Behalf Of Michael
> > >> Giacomelli
> > >> Sent: Tuesday, September 23, 2014 00:49
> > >> To: [hidden email]
> > >> Subject: Re: recommended distortion target for multiphoton?
> > >>
> > >> *****
> > >> To join, leave or search the confocal microscopy listserv, go to:
> > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> Post images on http://www.imgur.com and include the link in your
> posting.
> > >> *****
> > >>
> > >> Just to follow up, I contacted MicronLaser.com and got several 1 cm^2
> > >> grid target etched into borosilicate glass. They are ~20 um deep and
> > >> so I can flow different fluorophores through. The cost was very
> > >> reasonable, but they were machined with a CO2 laser, and so have some
> > >> thermal microcracking that makes them unsuitable as resolution
> > >> targets. Still, they are virtually indestructable (can park the beam
> > >> forever at one point and you'll never damage it), and the very large
> > >> size makes them quite handy for testing translation stages and
> mosaicing
> > software.
> > >>
> > >> I'd still like to find some who can do a nanosecond pulsed laser etch
> > >> and see about getting custom resolution targets cut, but overall, the
> > >> micronlaser parts were quite reasonable for what I needed.
> > >>
> > >> Mike
> > >>
> > >> On Wed, Jul 23, 2014 at 2:00 PM, Craig Brideau
> > >> <[hidden email]>
> > >> wrote:
> > >> > *****
> > >> > To join, leave or search the confocal microscopy listserv, go to:
> > >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> > Post images on http://www.imgur.com and include the link in your
> posting.
> > >> > *****
> > >> >
> > >> > I've been bugging various companies for years to get a good 2P and
> > >> > confocal fluoresence target developed, but thus far nobody's come
> > >> > up with anything that really works that costs less than $1000.
> > >> >
> > >> > Craig Brideau
> > >> >
> > >> >
> > >> > On Wed, Jul 23, 2014 at 11:26 AM, Michael Giacomelli <[hidden email]>
> > >> wrote:
> > >> >
> > >> >> *****
> > >> >> To join, leave or search the confocal microscopy listserv, go to:
> > >> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> >> Post images on http://www.imgur.com and include the link in your
> > posting.
> > >> >> *****
> > >> >>
> > >> >> Unfortunately, it is extremely difficult to record the reflection
> > >> >> mode image in our microscope, as the NIR light will not pass
> > >> >> through the dichroic, and even if we changed out the dichroic for
> > >> >> a beam splitter, out PMTs are not sensitive to NIR light.
> > >> >>
> > >> >> Translating a bead though is a very interesting idea. We use a
> > >> >> high precision thorlabs MLS stage. I will double check the
> > >> >> repeatability, but in theory I could program it to perform an
> > >> >> extremely high resolution distortion measurement. Of course,
> > >> >> getting a single bead onto a coverslip (or else programming
> > >> >> software to automatically identify the same bead across hundreds
> > >> >> of frames) will be somewhat tricky.
> > >> >>
> > >> >> Its a shame that getting targets for multiphoton is so difficult.
> > >> >> Ideal I suppose would be something etched in glass so that I could
> > >> >> flow my own fluorophore through it. I will have to look around
> > >> >> and see if I can get a lithography facility to make something like
> that.
> > >> >>
> > >> >> Thanks,
> > >> >> Mike
> > >> >>
> > >> >> On Wed, Jul 23, 2014 at 3:19 AM, Mark Cannell
> > >> >> <[hidden email]> wrote:
> > >> >> > *****
> > >> >> > To join, leave or search the confocal microscopy listserv, go to:
> > >> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> >> > Post images on http://www.imgur.com and include the link in your
> > >> >> posting.
> > >> >> > *****
> > >> >> >
> > >> >> > Thinking about this a bit more, I realised that your existing
> > >> >> > grid is
> > >> >> perfectly suitable as you don’t need 2P excitation to measure
> > >> >> field distortion at the wavelength you are using, Just turn the
> > >> >> power down a lot and record a reflection image,
> > >> >> >
> > >> >> > Cheers Mark
> > >> >> >
> > >> >> >>>
> > >> >> >>> Hi Mark,
> > >> >> >>>
> > >> >> >>> I'm assuming you mean beads deposited in a grid or something
> > similar?
> > >> >> >>> Could you point me to where you found this?
> > >> >> >>>
> > >> >> >>> Thanks,
> > >> >> >>> Mike
> > >> >> >>>
> > >> >> >>> On Tue, Jul 22, 2014 at 3:58 AM, Mark Cannell
> > >> >> >>> <[hidden email]> wrote:
> > >> >> >>>> *****
> > >> >> >>>> To join, leave or search the confocal microscopy listserv, go
> to:
> > >> >> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> >> >>>> Post images on http://www.imgur.com and include the link in
> > >> >> >>>> your
> > >> >> >>> posting.
> > >> >> >>>> *****
> > >> >> >>>>
> > >> >> >>>> Hi Michael
> > >> >> >>>>
> > >> >> >>>> I’ve always used beads to test the microscope. They are
> > >> >> >>>> relatively
> > >> >> cheap
> > >> >> >>> and if you blow one up it don’t matter. As you say, evaporated
> > >> >> >>> metal targets are too easily damaged (as Jim Pauley and I
> > >> >> >>> discovered when
> > >> >> testing
> > >> >> >>> my microscope back in ’95!)
> > >> >> >>>>
> > >> >> >>>> HTH
> > >> >> >>>>
> > >> >> >>>> Mark
> > >> >> >>>>
> > >> >> >>>> On 22/07/2014, at 4:30 am, Michael Giacomelli <[hidden email]>
> > >> wrote:
> > >> >> >>>>
> > >> >> >>>>> *****
> > >> >> >>>>> To join, leave or search the confocal microscopy listserv,
> go to:
> > >> >> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> >> >>>>> Post images on http://www.imgur.com and include the link in
> > >> >> >>>>> your
> > >> >> >>> posting.
> > >> >> >>>>> *****
> > >> >> >>>>>
> > >> >> >>>>> Could someone recommend a good multiphoton distortion target?
> > >> >> >>>>> Preferably one that is relatively robust against damage.
> > >> >> >>>>>
> > >> >> >>>>> Currently I am using a thorlabs wire grid target with
> > >> >> >>>>> fluorophore behind it. The beam is attenuated by the metal,
> > >> >> >>>>> resulting in an
> > >> >> image
> > >> >> >>>>> of the grid. However, passing through the entire 1 mm slide
> > >> >> >>>>> greatly reduces my resolution, and unless I am extremely
> > >> >> >>>>> careful, the grid is ablated by the beam.
> > >> >> >>>>>
> > >> >> >>>>> Thanks,
> > >> >> >>>>> Michael
> > >> >> >>>>
> > >> >> >>>>
> > >> >> >>>> Mark B. Cannell Ph.D. FRSNZ
> > >> >> >>>> Professor of Cardiac Cell Biology School of Physiology &
> > >> >> >>>> Pharmacology Medical Sciences Building University of Bristol
> > >> >> >>>> Bristol
> > >> >> >>>> BS8 1TD UK
> > >> >> >>>>
> > >> >> >>>> [hidden email]
> > >> >> >>>
> > >> >> >
> > >> >> >
> > >> >> >
> > >> >> > Mark B. Cannell Ph.D. FRSNZ
> > >> >> > Professor of Cardiac Cell Biology School of Physiology &
> > >> >> > Pharmacology Medical Sciences Building University of Bristol
> > >> >> > Bristol
> > >> >> > BS8 1TD UK
> > >> >> >
> > >> >> > [hidden email]
> > >> >>
>