dual ti-saphire multiphoton system

[Debora Keller]

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Hi confocalists

We are considering a dual ti-saphire option on our Leica SP5 used for intravitalimaging,
and being a total novice in the MP field, I was wondering if any of you had any experience
with that configuration.

At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with a l/2 and
polarizer to chose which one gets the MP) but we are going to purchase a second one.
The easy option is to separate both systems, so that both have a dedicated MP.
But another option is to have the possibility to use the 2 lasers on the intravital system
(upright SP5) to bypass the need of tuning between two wavelength (fairly slow) and
speed up acquisition.

Does anyone have experience with a dual-ti saphire system?
how do you setup the two lasers? do you use a 50-50 broadband plate beamspliter (to be
able to use the entire wavelength range, even if loosing 50% power) or a dichroic (in
which case one of the lasers could not be used below the cut-off wavelength?
What strategies do you have to combine the beams?

any help or comment will be appreciated!
Cheers

Debbi

Debora Keller, PhD

FILM - Facility for Imaging by Light Microscopy
- Super-Resolution Microscopy Specialist -
Sir Alexander Fleming Building, desk 408
Imperial College London / South Kensington
Exhibition Road
London SW7 2AZ, UK
Phone: [phone]
Mobile: + 44(0) 7760 256 889
E-mail:  [hidden email]
Website: http://imperial.ac.uk/imagingfacility

[Michael Giacomelli]

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On a lot of newer ti:saph lasers the power output is high enough that a
50:50 is fine.  Otherwise, I would recommend using a polarizing beam
splitter and an achromatized waveplate so that you can combine the two
lasers without power loss or wavelength sensitivity.  As an added bonus,
the waveplate can be used to tune the relative power of the second laser so
that one channel does not swamp the other as you tune on and off of the
crystal's gain peak.

Mike

On Wed, Nov 12, 2014 at 5:24 AM, Debora Keller <[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 confocalists
>
> We are considering a dual ti-saphire option on our Leica SP5 used for
> intravitalimaging,
> and being a total novice in the MP field, I was wondering if any of you
> had any experience
> with that configuration.
>
> At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with
> a l/2 and
> polarizer to chose which one gets the MP) but we are going to purchase a
> second one.
> The easy option is to separate both systems, so that both have a dedicated
> MP.
> But another option is to have the possibility to use the 2 lasers on the
> intravital system
> (upright SP5) to bypass the need of tuning between two wavelength (fairly
> slow) and
> speed up acquisition.
>
> Does anyone have experience with a dual-ti saphire system?
> how do you setup the two lasers? do you use a 50-50 broadband plate
> beamspliter (to be
> able to use the entire wavelength range, even if loosing 50% power) or a
> dichroic (in
> which case one of the lasers could not be used below the cut-off
> wavelength?
> What strategies do you have to combine the beams?
>
> any help or comment will be appreciated!
> Cheers
>
> Debbi
>
> Debora Keller, PhD
>
> FILM - Facility for Imaging by Light Microscopy
> - Super-Resolution Microscopy Specialist -
> Sir Alexander Fleming Building, desk 408
> Imperial College London / South Kensington
> Exhibition Road
> London SW7 2AZ, UK
> Phone: [phone]
> Mobile: + 44(0) 7760 256 889
> E-mail:  [hidden email]
> Website: http://imperial.ac.uk/imagingfacility
>

[Craig Brideau]

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Hi Debbi. I have a twin imaging OPA system that is basically the same thing
as two Ti:Saphs. I took a hard look at the pairs of dyes I would want to
excite simultaneously. I then selected a combiner dichroic coated for a
split point at 880nm. One beam can be tuned between 650-880nm, while the
other can be tuned from 880 to 1300nm (OPAs have a wider tuning range than
Ti:Saph). Once combined they are both sent to the microscope. My thinking
was I would typically be using GFP (likes 965nm approx) and then bluer dyes
requiring ~750nm. The 880 was a compromise. It is also possible to keep a
set of combiner dichroics on hand so you can select different split points
as necessary. Do keep in mind that it takes little bit of fiddling to
realign the system when you change dichroics, but if you keep a pair of
alignment irises in the system for each beam you can come back fairly
quickly.

Craig

On Wed, Nov 12, 2014 at 3:24 AM, Debora Keller <[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 confocalists
>
> We are considering a dual ti-saphire option on our Leica SP5 used for
> intravitalimaging,
> and being a total novice in the MP field, I was wondering if any of you
> had any experience
> with that configuration.
>
> At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with
> a l/2 and
> polarizer to chose which one gets the MP) but we are going to purchase a
> second one.
> The easy option is to separate both systems, so that both have a dedicated
> MP.
> But another option is to have the possibility to use the 2 lasers on the
> intravital system
> (upright SP5) to bypass the need of tuning between two wavelength (fairly
> slow) and
> speed up acquisition.
>
> Does anyone have experience with a dual-ti saphire system?
> how do you setup the two lasers? do you use a 50-50 broadband plate
> beamspliter (to be
> able to use the entire wavelength range, even if loosing 50% power) or a
> dichroic (in
> which case one of the lasers could not be used below the cut-off
> wavelength?
> What strategies do you have to combine the beams?
>
> any help or comment will be appreciated!
> Cheers
>
> Debbi
>
> Debora Keller, PhD
>
> FILM - Facility for Imaging by Light Microscopy
> - Super-Resolution Microscopy Specialist -
> Sir Alexander Fleming Building, desk 408
> Imperial College London / South Kensington
> Exhibition Road
> London SW7 2AZ, UK
> Phone: [phone]
> Mobile: + 44(0) 7760 256 889
> E-mail:  [hidden email]
> Website: http://imperial.ac.uk/imagingfacility
>

[Craig Brideau]

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*****

Do keep in mind that if you use a polarizing beam splitter to combine the
lasers then the lasers will have opposite polarizations (H vs. V) at the
sample plane. The advantage of the dichroic I proposed is that you don't
have to worry about this. This disadvantage is the wavelength range
limitations imparted by the split point, but conversely having opposing
polarization states for each laser may either be useful or a problem
depending on your sample and what you are exploring. From my own extensive
2P experience I have discovered that ordered samples are strongly
influenced by the polarization of the excitation light.

Craig Brideau

On Wed, Nov 12, 2014 at 10:00 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.
> *****
>
> On a lot of newer ti:saph lasers the power output is high enough that a
> 50:50 is fine.  Otherwise, I would recommend using a polarizing beam
> splitter and an achromatized waveplate so that you can combine the two
> lasers without power loss or wavelength sensitivity.  As an added bonus,
> the waveplate can be used to tune the relative power of the second laser so
> that one channel does not swamp the other as you tune on and off of the
> crystal's gain peak.
>
> Mike
>
> On Wed, Nov 12, 2014 at 5:24 AM, Debora Keller <[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 confocalists
> >
> > We are considering a dual ti-saphire option on our Leica SP5 used for
> > intravitalimaging,
> > and being a total novice in the MP field, I was wondering if any of you
> > had any experience
> > with that configuration.
> >
> > At the moment our two SP5s are sharing one Mai Tai multiphoton laser
> (with
> > a l/2 and
> > polarizer to chose which one gets the MP) but we are going to purchase a
> > second one.
> > The easy option is to separate both systems, so that both have a
> dedicated
> > MP.
> > But another option is to have the possibility to use the 2 lasers on the
> > intravital system
> > (upright SP5) to bypass the need of tuning between two wavelength (fairly
> > slow) and
> > speed up acquisition.
> >
> > Does anyone have experience with a dual-ti saphire system?
> > how do you setup the two lasers? do you use a 50-50 broadband plate
> > beamspliter (to be
> > able to use the entire wavelength range, even if loosing 50% power) or a
> > dichroic (in
> > which case one of the lasers could not be used below the cut-off
> > wavelength?
> > What strategies do you have to combine the beams?
> >
> > any help or comment will be appreciated!
> > Cheers
> >
> > Debbi
> >
> > Debora Keller, PhD
> >
> > FILM - Facility for Imaging by Light Microscopy
> > - Super-Resolution Microscopy Specialist -
> > Sir Alexander Fleming Building, desk 408
> > Imperial College London / South Kensington
> > Exhibition Road
> > London SW7 2AZ, UK
> > Phone: [phone]
> > Mobile: + 44(0) 7760 256 889
> > E-mail:  [hidden email]
> > Website: http://imperial.ac.uk/imagingfacility
> >
>

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

Hi Debbi,

I have been setting up multiple tales where systems share two or more lasers. What we usually do is that split the laser beam with polarization beam splitter cube and then pass beam over the Pockel cells before combine them together with polarization beam splitter cube. This configuration has one advantage that any of your lasers can be used for imaging and they have their own power control.

Long

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Debora Keller
Sent: Wednesday, November 12, 2014 5:25 AM
To: [hidden email]
Subject: dual ti-saphire multiphoton system

*****
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 confocalists

We are considering a dual ti-saphire option on our Leica SP5 used for intravitalimaging, and being a total novice in the MP field, I was wondering if any of you had any experience with that configuration.

At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with a l/2 and polarizer to chose which one gets the MP) but we are going to purchase a second one.
The easy option is to separate both systems, so that both have a dedicated MP.
But another option is to have the possibility to use the 2 lasers on the intravital system (upright SP5) to bypass the need of tuning between two wavelength (fairly slow) and speed up acquisition.

Does anyone have experience with a dual-ti saphire system?
how do you setup the two lasers? do you use a 50-50 broadband plate beamspliter (to be able to use the entire wavelength range, even if loosing 50% power) or a dichroic (in which case one of the lasers could not be used below the cut-off wavelength?
What strategies do you have to combine the beams?

any help or comment will be appreciated!
Cheers

Debbi

Debora Keller, PhD

FILM - Facility for Imaging by Light Microscopy
- Super-Resolution Microscopy Specialist - Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ, UK
Phone: [phone]
Mobile: + 44(0) 7760 256 889
E-mail:  [hidden email]
Website: http://imperial.ac.uk/imagingfacility

[Shalin Mehta]

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Hi Debbie,
The achromatic nature/power preservation of polarizing beam
splitter/combiner is a plus. But as Craig points out, samples such as
FM dye labeled lipid membrane, phalloidin labeled actin, or taxol
labeled microtubules will light up differently depending on
polarization of excitation laser since the dipoles of fluorophores are
strongly aligned. If your MP dichroic highly distorts polarization,
you don't have to worry about this, because the linear polarization
will become ellpitical.

If you have polarization preserving dichroic (something I have had
hard time finding), you could add a waveplate (lambda/4 not lambda/2)
at 45 degrees to both of your lasers (horizontal and vertically
polarized). This will convert both beams into circular polarization of
opposite handedness.

If you go this route, the thing to do will be to take some cells with
strong stress fiber labeled with phalloidin-Alexa Fluor 488. Excite it
with your MP laser and see if you notice any orientation-dependent
signal (rotate the dish). If you do, add an IR lambda/4 plate and
rotate the plate until the bias disappears.

A shameless plug next: we make use of polarization of excitation to
look at orientation of structures
(http://www.focusonmicroscopy.org/2014/PDF/200_Mehta.pdf).

All the best
Shalin
Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.


On Wed, Nov 12, 2014 at 1:43 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.
> *****
>
> Do keep in mind that if you use a polarizing beam splitter to combine the
> lasers then the lasers will have opposite polarizations (H vs. V) at the
> sample plane. The advantage of the dichroic I proposed is that you don't
> have to worry about this. This disadvantage is the wavelength range
> limitations imparted by the split point, but conversely having opposing
> polarization states for each laser may either be useful or a problem
> depending on your sample and what you are exploring. From my own extensive
> 2P experience I have discovered that ordered samples are strongly
> influenced by the polarization of the excitation light.
>
> Craig Brideau
>
> On Wed, Nov 12, 2014 at 10:00 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.
>> *****
>>
>> On a lot of newer ti:saph lasers the power output is high enough that a
>> 50:50 is fine.  Otherwise, I would recommend using a polarizing beam
>> splitter and an achromatized waveplate so that you can combine the two
>> lasers without power loss or wavelength sensitivity.  As an added bonus,
>> the waveplate can be used to tune the relative power of the second laser so
>> that one channel does not swamp the other as you tune on and off of the
>> crystal's gain peak.
>>
>> Mike
>>
>> On Wed, Nov 12, 2014 at 5:24 AM, Debora Keller <[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 confocalists
>> >
>> > We are considering a dual ti-saphire option on our Leica SP5 used for
>> > intravitalimaging,
>> > and being a total novice in the MP field, I was wondering if any of you
>> > had any experience
>> > with that configuration.
>> >
>> > At the moment our two SP5s are sharing one Mai Tai multiphoton laser
>> (with
>> > a l/2 and
>> > polarizer to chose which one gets the MP) but we are going to purchase a
>> > second one.
>> > The easy option is to separate both systems, so that both have a
>> dedicated
>> > MP.
>> > But another option is to have the possibility to use the 2 lasers on the
>> > intravital system
>> > (upright SP5) to bypass the need of tuning between two wavelength (fairly
>> > slow) and
>> > speed up acquisition.
>> >
>> > Does anyone have experience with a dual-ti saphire system?
>> > how do you setup the two lasers? do you use a 50-50 broadband plate
>> > beamspliter (to be
>> > able to use the entire wavelength range, even if loosing 50% power) or a
>> > dichroic (in
>> > which case one of the lasers could not be used below the cut-off
>> > wavelength?
>> > What strategies do you have to combine the beams?
>> >
>> > any help or comment will be appreciated!
>> > Cheers
>> >
>> > Debbi
>> >
>> > Debora Keller, PhD
>> >
>> > FILM - Facility for Imaging by Light Microscopy
>> > - Super-Resolution Microscopy Specialist -
>> > Sir Alexander Fleming Building, desk 408
>> > Imperial College London / South Kensington
>> > Exhibition Road
>> > London SW7 2AZ, UK
>> > Phone: [phone]
>> > Mobile: + 44(0) 7760 256 889
>> > E-mail:  [hidden email]
>> > Website: http://imperial.ac.uk/imagingfacility
>> >
>>

[mcammer]

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An investigator here has a dual laser system.  But I wonder if we are pumping too much IR in at once and if it would be better to have a system that could alternate the pulses or have the lasers intentionally out of synch by exactly half a period.

===========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   note that we do not receive messages left at 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Wednesday, November 12, 2014 1:55 PM
To: [hidden email]
Subject: Re: dual ti-saphire multiphoton system

*****
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*****

Hi Debbi,

I have been setting up multiple tales where systems share two or more lasers. What we usually do is that split the laser beam with polarization beam splitter cube and then pass beam over the Pockel cells before combine them together with polarization beam splitter cube. This configuration has one advantage that any of your lasers can be used for imaging and they have their own power control.

Long

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Debora Keller
Sent: Wednesday, November 12, 2014 5:25 AM
To: [hidden email]
Subject: dual ti-saphire multiphoton system

*****
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 confocalists

We are considering a dual ti-saphire option on our Leica SP5 used for intravitalimaging, and being a total novice in the MP field, I was wondering if any of you had any experience with that configuration.

At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with a l/2 and polarizer to chose which one gets the MP) but we are going to purchase a second one.
The easy option is to separate both systems, so that both have a dedicated MP.
But another option is to have the possibility to use the 2 lasers on the intravital system (upright SP5) to bypass the need of tuning between two wavelength (fairly slow) and speed up acquisition.

Does anyone have experience with a dual-ti saphire system?
how do you setup the two lasers? do you use a 50-50 broadband plate beamspliter (to be able to use the entire wavelength range, even if loosing 50% power) or a dichroic (in which case one of the lasers could not be used below the cut-off wavelength?
What strategies do you have to combine the beams?

any help or comment will be appreciated!
Cheers

Debbi

Debora Keller, PhD

FILM - Facility for Imaging by Light Microscopy
- Super-Resolution Microscopy Specialist - Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ, UK
Phone: [phone]
Mobile: + 44(0) 7760 256 889
E-mail:  [hidden email]
Website: http://imperial.ac.uk/imagingfacility

[Craig Brideau]

*****
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*****

Well, if the two Ti:Saphs are unsynchronized, then the spacing between the
pulses will be random. Their repetition rates will probably be slightly
different, so you will get frequency beating somewhat. Most of the time
though their pulses should not be temporally coincident.

Craig

On Wed, Nov 12, 2014 at 1:40 PM, Cammer, Michael <[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.
> *****
>
> An investigator here has a dual laser system.  But I wonder if we are
> pumping too much IR in at once and if it would be better to have a system
> that could alternate the pulses or have the lasers intentionally out of
> synch by exactly half a period.
>
> ===========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical
> Center
> Cell:  914-309-3270   note that we do not receive messages left at
> 212-263-3208
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of [hidden email]
> Sent: Wednesday, November 12, 2014 1:55 PM
> To: [hidden email]
> Subject: Re: dual ti-saphire multiphoton system
>
> *****
> 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 Debbi,
>
> I have been setting up multiple tales where systems share two or more
> lasers. What we usually do is that split the laser beam with polarization
> beam splitter cube and then pass beam over the Pockel cells before combine
> them together with polarization beam splitter cube. This configuration has
> one advantage that any of your lasers can be used for imaging and they have
> their own power control.
>
> Long
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Debora Keller
> Sent: Wednesday, November 12, 2014 5:25 AM
> To: [hidden email]
> Subject: dual ti-saphire multiphoton system
>
> *****
> 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 confocalists
>
> We are considering a dual ti-saphire option on our Leica SP5 used for
> intravitalimaging, and being a total novice in the MP field, I was
> wondering if any of you had any experience with that configuration.
>
> At the moment our two SP5s are sharing one Mai Tai multiphoton laser (with
> a l/2 and polarizer to chose which one gets the MP) but we are going to
> purchase a second one.
> The easy option is to separate both systems, so that both have a dedicated
> MP.
> But another option is to have the possibility to use the 2 lasers on the
> intravital system (upright SP5) to bypass the need of tuning between two
> wavelength (fairly slow) and speed up acquisition.
>
> Does anyone have experience with a dual-ti saphire system?
> how do you setup the two lasers? do you use a 50-50 broadband plate
> beamspliter (to be able to use the entire wavelength range, even if loosing
> 50% power) or a dichroic (in which case one of the lasers could not be used
> below the cut-off wavelength?
> What strategies do you have to combine the beams?
>
> any help or comment will be appreciated!
> Cheers
>
> Debbi
>
> Debora Keller, PhD
>
> FILM - Facility for Imaging by Light Microscopy
> - Super-Resolution Microscopy Specialist - Sir Alexander Fleming Building,
> desk 408 Imperial College London / South Kensington Exhibition Road London
> SW7 2AZ, UK
> Phone: [phone]
> Mobile: + 44(0) 7760 256 889
> E-mail:  [hidden email]
> Website: http://imperial.ac.uk/imagingfacility
>

[Michael Giacomelli]

*****
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*****

The pulse rate of even the same ti:saph changes quite a lot with wavelength
when tuning, so getting two of them to pulse in phase is quite expensive.
Coherent will sell you a device that can do it on some of their lasers (the
Mira has one for somewhere in the five figures if I recall correctly) by
dynamically controlling the cavity length using a PZT and a very high
bandwidth PLL.  We have a home built version in our lab here, but it is
quite painful to configure and we have not run it in some years.

Alternatively you can buy an OPO which will give you two synchronized
pulses at any wavelength you like, but that will make even a Ti:Saph look
cheap. Fortunately, unless you are doing pump probe imaging or similar
technique, there is usually no need to synchronize pulses.  A simple beam
splitter or dichroic will work fine for a tiny fraction of the cost.

Mike

On Wed, Nov 12, 2014 at 4:26 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.
> *****
>
> Well, if the two Ti:Saphs are unsynchronized, then the spacing between the
> pulses will be random. Their repetition rates will probably be slightly
> different, so you will get frequency beating somewhat. Most of the time
> though their pulses should not be temporally coincident.
>
> Craig
>
> On Wed, Nov 12, 2014 at 1:40 PM, Cammer, Michael <
> [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.
> > *****
> >
> > An investigator here has a dual laser system.  But I wonder if we are
> > pumping too much IR in at once and if it would be better to have a system
> > that could alternate the pulses or have the lasers intentionally out of
> > synch by exactly half a period.
> >
> >
> ===========================================================================
> > Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical
> > Center
> > Cell:  914-309-3270   note that we do not receive messages left at
> > 212-263-3208
> > http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of [hidden email]
> > Sent: Wednesday, November 12, 2014 1:55 PM
> > To: [hidden email]
> > Subject: Re: dual ti-saphire multiphoton system
> >
> > *****
> > 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 Debbi,
> >
> > I have been setting up multiple tales where systems share two or more
> > lasers. What we usually do is that split the laser beam with polarization
> > beam splitter cube and then pass beam over the Pockel cells before
> combine
> > them together with polarization beam splitter cube. This configuration
> has
> > one advantage that any of your lasers can be used for imaging and they
> have
> > their own power control.
> >
> > Long
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Debora Keller
> > Sent: Wednesday, November 12, 2014 5:25 AM
> > To: [hidden email]
> > Subject: dual ti-saphire multiphoton system
> >
> > *****
> > 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 confocalists
> >
> > We are considering a dual ti-saphire option on our Leica SP5 used for
> > intravitalimaging, and being a total novice in the MP field, I was
> > wondering if any of you had any experience with that configuration.
> >
> > At the moment our two SP5s are sharing one Mai Tai multiphoton laser
> (with
> > a l/2 and polarizer to chose which one gets the MP) but we are going to
> > purchase a second one.
> > The easy option is to separate both systems, so that both have a
> dedicated
> > MP.
> > But another option is to have the possibility to use the 2 lasers on the
> > intravital system (upright SP5) to bypass the need of tuning between two
> > wavelength (fairly slow) and speed up acquisition.
> >
> > Does anyone have experience with a dual-ti saphire system?
> > how do you setup the two lasers? do you use a 50-50 broadband plate
> > beamspliter (to be able to use the entire wavelength range, even if
> loosing
> > 50% power) or a dichroic (in which case one of the lasers could not be
> used
> > below the cut-off wavelength?
> > What strategies do you have to combine the beams?
> >
> > any help or comment will be appreciated!
> > Cheers
> >
> > Debbi
> >
> > Debora Keller, PhD
> >
> > FILM - Facility for Imaging by Light Microscopy
> > - Super-Resolution Microscopy Specialist - Sir Alexander Fleming
> Building,
> > desk 408 Imperial College London / South Kensington Exhibition Road
> London
> > SW7 2AZ, UK
> > Phone: [phone]
> > Mobile: + 44(0) 7760 256 889
> > E-mail:  [hidden email]
> > Website: http://imperial.ac.uk/imagingfacility
> >
>

[Kate Luby-Phelps]

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*****

We set ours up to do it both ways. Don't know if that is an option with the Leica.

Kate Luby-Phelps
Director, UT Southwestern Live Cell Imaging Facility
Dallas, TX

[Debora Keller]

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Dear Listers,

Thanks a lot for all the input - I have now quite some food for thoughts!

I was not aware of the polarization issues, but will keep this in mind (though the impact
on the final image seems to be dependant on the structure observed).

At least now I have a couple of arguments/points to discuss with Leica and decide what is
doable with our setups.
Cheers

Debbi

PS: obviously, any additional comments will still be read with high interest!

Debora Keller, PhD

FILM - Facility for Imaging by Light Microscopy
- Super-Resolution Microscopy Specialist -
Sir Alexander Fleming Building, desk 408
Imperial College London / South Kensington
Exhibition Road
London SW7 2AZ, UK
Phone: [phone]
Mobile: + 44(0) 7760 256 889
E-mail:  [hidden email]
Website: http://imperial.ac.uk/imagingfacility

[Michael Giacomelli]

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*****

Hi Debora,

Could you clarify if you are actually power limited?  If this is something
like a 4 watt Chameleon and you are doing tissue imaging at 0.05 watts
power, than most of the discussion is irrelevant.  A simple 50/50
beamsplitter from thorlabs will combine your laser with no effort over the
entire tuning range while still giving you 40 times more power than you
actually need.

Mike



On Thu, Nov 13, 2014 at 12:08 PM, Debora Keller <[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.
> *****
>
> Dear Listers,
>
> Thanks a lot for all the input - I have now quite some food for thoughts!
>
> I was not aware of the polarization issues, but will keep this in mind
> (though the impact
> on the final image seems to be dependant on the structure observed).
>
> At least now I have a couple of arguments/points to discuss with Leica and
> decide what is
> doable with our setups.
> Cheers
>
> Debbi
>
> PS: obviously, any additional comments will still be read with high
> interest!
>
> Debora Keller, PhD
>
> FILM - Facility for Imaging by Light Microscopy
> - Super-Resolution Microscopy Specialist -
> Sir Alexander Fleming Building, desk 408
> Imperial College London / South Kensington
> Exhibition Road
> London SW7 2AZ, UK
> Phone: [phone]
> Mobile: + 44(0) 7760 256 889
> E-mail:  [hidden email]
> Website: http://imperial.ac.uk/imagingfacility
>

[Debora Keller]

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

Hi Mike,

I am unsure: we will be getting the new eHP MaiTai DeepSee specified with an average
power of >2.4W. We would combine it with an "old" Mai Tai (potentially upgraded with a
DeepSee module) which currently gives us ~ 2W at 860nm but should be a bit higher
(engineers have to re-align it slightly).

I am not sure if this counts as power-limitation or not, does it?
For the current imaging, the lasers are used at ~ 6% - 12.5% of power (via Leica's EOM),
so we could use higher % in the case of a 50/50.
Cheers

Debbi


On Thu, 13 Nov 2014 12:16:48 -0500, 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 Debora,
>
>Could you clarify if you are actually power limited?  If this is something
>like a 4 watt Chameleon and you are doing tissue imaging at 0.05 watts
>power, than most of the discussion is irrelevant.  A simple 50/50
>beamsplitter from thorlabs will combine your laser with no effort over the
>entire tuning range while still giving you 40 times more power than you
>actually need.
>
>Mike
>
>
>
>On Thu, Nov 13, 2014 at 12:08 PM, Debora Keller <[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.
>> *****
>>
>> Dear Listers,
>>
>> Thanks a lot for all the input - I have now quite some food for thoughts!
>>
>> I was not aware of the polarization issues, but will keep this in mind
>> (though the impact
>> on the final image seems to be dependant on the structure observed).
>>
>> At least now I have a couple of arguments/points to discuss with Leica and
>> decide what is
>> doable with our setups.
>> Cheers
>>
>> Debbi
>>
>> PS: obviously, any additional comments will still be read with high
>> interest!
>>
>> Debora Keller, PhD
>>
>> FILM - Facility for Imaging by Light Microscopy
>> - Super-Resolution Microscopy Specialist -
>> Sir Alexander Fleming Building, desk 408
>> Imperial College London / South Kensington
>> Exhibition Road
>> London SW7 2AZ, UK
>> Phone: [phone]
>> Mobile: + 44(0) 7760 256 889
>> E-mail:  [hidden email]
>> Website: http://imperial.ac.uk/imagingfacility
>>

[Michael Giacomelli]

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

Hi Debbi,

2 W per beam is quite a lot of power.  I've burned up samples at a fraction
of that power.  I would think a simple beam splitter is fine for your
application.  If you were using 500 mW TiSaphs I would recommend trying to
conserve power.

Mike

On Fri, Nov 14, 2014 at 8:29 AM, Debora Keller <[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 Mike,
>
> I am unsure: we will be getting the new eHP MaiTai DeepSee specified with
> an average
> power of >2.4W. We would combine it with an "old" Mai Tai (potentially
> upgraded with a
> DeepSee module) which currently gives us ~ 2W at 860nm but should be a bit
> higher
> (engineers have to re-align it slightly).
>
> I am not sure if this counts as power-limitation or not, does it?
> For the current imaging, the lasers are used at ~ 6% - 12.5% of power (via
> Leica's EOM),
> so we could use higher % in the case of a 50/50.
> Cheers
>
> Debbi
>
>
> On Thu, 13 Nov 2014 12:16:48 -0500, 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 Debora,
> >
> >Could you clarify if you are actually power limited?  If this is something
> >like a 4 watt Chameleon and you are doing tissue imaging at 0.05 watts
> >power, than most of the discussion is irrelevant.  A simple 50/50
> >beamsplitter from thorlabs will combine your laser with no effort over the
> >entire tuning range while still giving you 40 times more power than you
> >actually need.
> >
> >Mike
> >
> >
> >
> >On Thu, Nov 13, 2014 at 12:08 PM, Debora Keller <[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.
> >> *****
> >>
> >> Dear Listers,
> >>
> >> Thanks a lot for all the input - I have now quite some food for
> thoughts!
> >>
> >> I was not aware of the polarization issues, but will keep this in mind
> >> (though the impact
> >> on the final image seems to be dependant on the structure observed).
> >>
> >> At least now I have a couple of arguments/points to discuss with Leica
> and
> >> decide what is
> >> doable with our setups.
> >> Cheers
> >>
> >> Debbi
> >>
> >> PS: obviously, any additional comments will still be read with high
> >> interest!
> >>
> >> Debora Keller, PhD
> >>
> >> FILM - Facility for Imaging by Light Microscopy
> >> - Super-Resolution Microscopy Specialist -
> >> Sir Alexander Fleming Building, desk 408
> >> Imperial College London / South Kensington
> >> Exhibition Road
> >> London SW7 2AZ, UK
> >> Phone: [phone]
> >> Mobile: + 44(0) 7760 256 889
> >> E-mail:  [hidden email]
> >> Website: http://imperial.ac.uk/imagingfacility
> >>
>