laser choices for multiphoton

[mcammer]

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We are considering a multiphoton purchase and a big issue is illumination.  Putting budget constraints aside and thinking purely of imaging non-destructively in live tissues, what would give us the most flexibility for exciting multiple probes for maximum signal while minimally damaging the biology?



We are used to using lasers that tune from approx 700 to 1060 nm and mostly use 890 to 930 nm, but this does not provide good red imaging.  If money were no issue, would we be wise to get a laser such as the dual line Insight or two lasers from 690 to 1080 nm?



Some of the questions that have come up are:

*         We think a laser that tunesup to 1300 nm would solve the red imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of collagen would we need to use a different wavelength?  If so, how long does this take and do the commercial systems support this?

*         With a dual line laser that tunes out to 1300 nm is the fixed 1046 nm line really useful?

*         Does a dual line system cook the sample?



Any thoughts on this (and on specific multiphoton scopes) greatly appreciated.



Thank you!


=========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270     Office: Skirball 2nd Floor main office
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


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[George McNamara]

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Hi Michael,

How about Coherent Chameleon OPO-Vis or MPX? (or equivalent from other
vendors).

https://www.coherent.com/products/?1642/Chameleon-Compact-OPO-Family

*New:  Chameleon MPX* extends the wavelength range of Chameleon Vision
and Ultra Ti:Sapphire lasers and is specifically designed and optimized
for non-linear imaging techniques.

Employing is the latest generation fan-poled OPO technology; the fully
automated Chameleon MPX delivers high peak power to the sample plane
with short pulse pulses and dispersion compensation optimized for
typical commercial microscope systems.

Featuring a wide pump tuning range, the Chameleon MPX offers
independently tunable dual beam excitation of popular fluorescent
markers (eg. eGFP, mCherry), enabling powerful and truly flexible
multimodal imaging

*Chameleon Compact OPO*  Long wavelength tuning capability up to 1600
nm, with idler option to extend to 4000 nm. Designed for simplicity and
ease-of-use by non-laser experts, Compact OPO is a truly “black box”
wavelength extension. No adjustment is required during tuning and the
OPO can be controlled via USB or RS232 link.
*Chameleon Compact OPO-Vis* provides automated frequency doubling of the
OPO and laser output..  Used in conjunction with the Compact OPO and
Chameleon pump lasers, it gives access to an unprecedented automated
tuning range of 340 nm to 1600 nm, with simple touch screen controls and
no manual intervention. This OPO provides hundreds of milliwatts of
output power over most of its tuning range that can be expanded to cover
also the mid IR region between 1750 nm and 4000 nm.

The visible wavelengths could be used at low power for 1p excitation
down to 340 nm (no need for vis lasers), less phototoxicity, either for
(examples):
* Fura-2 (+Ca++)
* Brilliant Ultraviolet ... and ~395 nm for Brilliant Violet's (and
longer wavelengths for Brilliant Blue's)
* NIRvana Sciences chlorins ... http://nirvanasciences.com/?page_id=3088
* NADH, NADPH (see below).

MPEF may be better for NADH, and with a new fluorescent protein
biosensor for NAD+ along with existing NADH FP biosensors, direct
excitation of NADH may not matter any more (since the FP's may be
brighter and can localize to a compartment of interest).


There is potential to shape the beam(s) to get 2 or more STED doughnuts
at different wavelengths (and I think the plate can go where the DIC
Wollaston prism would go). See

https://works.bepress.com/gmcnamara/3/
https://works.bepress.com/gmcnamara/13/

... there is a newer Q-plate for STED

https://www.osapublishing.org/abstract.cfm?uri=cleo_si-2015-STu1L.5

//

Chris Xu (Cornell Univ) has published on using very long wavelengths

http://www.ncbi.nlm.nih.gov/pubmed/25909022 ... 1700 nm

http://www.ncbi.nlm.nih.gov/pubmed/25360361 ... 2p, 3p, 4p using 800,
1300 and 1680 nm


best wishes,

George


On 7/7/2016 11:54 AM, Cammer, Michael 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.
> *****
>
> We are considering a multiphoton purchase and a big issue is illumination.  Putting budget constraints aside and thinking purely of imaging non-destructively in live tissues, what would give us the most flexibility for exciting multiple probes for maximum signal while minimally damaging the biology?
>
>
>
> We are used to using lasers that tune from approx 700 to 1060 nm and mostly use 890 to 930 nm, but this does not provide good red imaging.  If money were no issue, would we be wise to get a laser such as the dual line Insight or two lasers from 690 to 1080 nm?
>
>
>
> Some of the questions that have come up are:
>
> *         We think a laser that tunesup to 1300 nm would solve the red imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of collagen would we need to use a different wavelength?  If so, how long does this take and do the commercial systems support this?
>
> *         With a dual line laser that tunes out to 1300 nm is the fixed 1046 nm line really useful?
>
> *         Does a dual line system cook the sample?
>
>
>
> Any thoughts on this (and on specific multiphoton scopes) greatly appreciated.
>
>
>
> Thank you!
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell:  914-309-3270     Office: Skirball 2nd Floor main office
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/

[Steffen Dietzel]

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Michael,

you probably are aware that some red dyes do just nicely when excited
around 860 or so, although is most likely not the S1-state (Mütze et
al., Excitation Spectra and Brightness Optimization of Two-Photon
Excited Probes. doi:10.1016/j.bpj.2011.12.056)

Having said that, I like the possibility to tune up to 1300 nm. It gives
you much more flexibility. e.g. exciting Eosin is not a problem, or the
red FPs. Also I like THG a lot, usually around 1275 nm excitation, with
the THG signal at 425 nm and SHG at 638.

So far we didn't have an application where we needed a high laser line
(e.g. 1200) and a low one (850) simultaneously. If one comes around we
would have to tune the laser, which takes time. If this is a frequent
request at your site, you might be better of with two lasers. If money
were no issue, I'd suggest two lasers tunable up to 1300nm...

Our old LaVision BioTech and also our new Leica SP8 both support
sequential scanning with several wavelengths (tuning in-between), I
suppose the others do too.

I believe the fixed 1046 is useful only if you want to do CARS. Or if
you happen to have fluors that are nicely excited at that range. But I
don't know any. In that respect a TiSa-OPO combination may be more
useful were you can take out 10% of a 4 W laser to image directly
(e.g.834 nm) and the 90% to generate a long wavelength.

I wouldn't worry too much about cooking. One, the absorption of tissue
is very low between 1200 and 1300. Two, the transmittance of the
microscopes is very low, so you might get not so much out of the
objective (still plenty to do fluorescence). Three, if you are still
worried, a resonant scanner might be a good idea.

Steffen


Am 07.07.2016 um 18:54 schrieb Cammer, Michael:

> *****
> 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.
> *****
>
> We are considering a multiphoton purchase and a big issue is illumination.  Putting budget constraints aside and thinking purely of imaging non-destructively in live tissues, what would give us the most flexibility for exciting multiple probes for maximum signal while minimally damaging the biology?
>
>
>
> We are used to using lasers that tune from approx 700 to 1060 nm and mostly use 890 to 930 nm, but this does not provide good red imaging.  If money were no issue, would we be wise to get a laser such as the dual line Insight or two lasers from 690 to 1080 nm?
>
>
>
> Some of the questions that have come up are:
>
> *         We think a laser that tunesup to 1300 nm would solve the red imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of collagen would we need to use a different wavelength?  If so, how long does this take and do the commercial systems support this?
>
> *         With a dual line laser that tunes out to 1300 nm is the fixed 1046 nm line really useful?
>
> *         Does a dual line system cook the sample?
>
>
>
> Any thoughts on this (and on specific multiphoton scopes) greatly appreciated.
>
>
>
> Thank you!
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell:  914-309-3270     Office: Skirball 2nd Floor main office
> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Craig Brideau]

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*****

As Steffen mentions, you can sometimes excite red dyes with surprisingly
short wavelengths in 2p. When this happens you are generally exciting an S2
or higher state which can give mixed results. Another possibility is 2+1
photon, where you 2-photon excite to an intermediate but generally
non-radiative level, then have a third photon arrive to bump it up a little
further into a state from which it can radiate. Note this is not three
photon, the difference being whether virtual or real energy states are
involved in the transitions.

Craig

On Fri, Jul 8, 2016 at 8:14 AM, Steffen Dietzel <[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.
> *****
>
> Michael,
>
> you probably are aware that some red dyes do just nicely when excited
> around 860 or so, although is most likely not the S1-state (Mütze et al.,
> Excitation Spectra and Brightness Optimization of Two-Photon Excited
> Probes. doi:10.1016/j.bpj.2011.12.056)
>
> Having said that, I like the possibility to tune up to 1300 nm. It gives
> you much more flexibility. e.g. exciting Eosin is not a problem, or the red
> FPs. Also I like THG a lot, usually around 1275 nm excitation, with the THG
> signal at 425 nm and SHG at 638.
>
> So far we didn't have an application where we needed a high laser line
> (e.g. 1200) and a low one (850) simultaneously. If one comes around we
> would have to tune the laser, which takes time. If this is a frequent
> request at your site, you might be better of with two lasers. If money were
> no issue, I'd suggest two lasers tunable up to 1300nm...
>
> Our old LaVision BioTech and also our new Leica SP8 both support
> sequential scanning with several wavelengths (tuning in-between), I suppose
> the others do too.
>
> I believe the fixed 1046 is useful only if you want to do CARS. Or if you
> happen to have fluors that are nicely excited at that range. But I don't
> know any. In that respect a TiSa-OPO combination may be more useful were
> you can take out 10% of a 4 W laser to image directly (e.g.834 nm) and the
> 90% to generate a long wavelength.
>
> I wouldn't worry too much about cooking. One, the absorption of tissue is
> very low between 1200 and 1300. Two, the transmittance of the microscopes
> is very low, so you might get not so much out of the objective (still
> plenty to do fluorescence). Three, if you are still worried, a resonant
> scanner might be a good idea.
>
> Steffen
>
>
>
> Am 07.07.2016 um 18:54 schrieb Cammer, Michael:
>
>> *****
>> 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.
>> *****
>>
>> We are considering a multiphoton purchase and a big issue is
>> illumination.  Putting budget constraints aside and thinking purely of
>> imaging non-destructively in live tissues, what would give us the most
>> flexibility for exciting multiple probes for maximum signal while minimally
>> damaging the biology?
>>
>>
>>
>> We are used to using lasers that tune from approx 700 to 1060 nm and
>> mostly use 890 to 930 nm, but this does not provide good red imaging.  If
>> money were no issue, would we be wise to get a laser such as the dual line
>> Insight or two lasers from 690 to 1080 nm?
>>
>>
>>
>> Some of the questions that have come up are:
>>
>> *         We think a laser that tunesup to 1300 nm would solve the red
>> imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of
>> collagen would we need to use a different wavelength?  If so, how long does
>> this take and do the commercial systems support this?
>>
>> *         With a dual line laser that tunes out to 1300 nm is the fixed
>> 1046 nm line really useful?
>>
>> *         Does a dual line system cook the sample?
>>
>>
>>
>> Any thoughts on this (and on specific multiphoton scopes) greatly
>> appreciated.
>>
>>
>>
>> Thank you!
>>
>>
>> =========================================================================
>> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical
>> Center
>> Cell:  914-309-3270     Office: Skirball 2nd Floor main office
>> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>>
>>
>> ------------------------------------------------------------
>> This email message, including any attachments, is for the sole use of the
>> intended recipient(s) and may contain information that is proprietary,
>> confidential, and exempt from disclosure under applicable law. Any
>> unauthorized review, use, disclosure, or distribution is prohibited. If you
>> have received this email in error please notify the sender by return email
>> and delete the original message. Please note, the recipient should check
>> this email and any attachments for the presence of viruses. The
>> organization accepts no liability for any damage caused by any virus
>> transmitted by this email.
>> =================================
>>
>>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

[Dr. K N Ganesh]

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*****

Try some of the IR corrected silicone immersion objectives (Super apo) They
have good transmission range upto 1600nm.
No commercial interests please.
Reg
Ganesh
On Jul 8, 2016 7:45 PM, "Steffen Dietzel" <[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.
> *****
>
> Michael,
>
> you probably are aware that some red dyes do just nicely when excited
> around 860 or so, although is most likely not the S1-state (Mütze et al.,
> Excitation Spectra and Brightness Optimization of Two-Photon Excited
> Probes. doi:10.1016/j.bpj.2011.12.056)
>
> Having said that, I like the possibility to tune up to 1300 nm. It gives
> you much more flexibility. e.g. exciting Eosin is not a problem, or the red
> FPs. Also I like THG a lot, usually around 1275 nm excitation, with the THG
> signal at 425 nm and SHG at 638.
>
> So far we didn't have an application where we needed a high laser line
> (e.g. 1200) and a low one (850) simultaneously. If one comes around we
> would have to tune the laser, which takes time. If this is a frequent
> request at your site, you might be better of with two lasers. If money were
> no issue, I'd suggest two lasers tunable up to 1300nm...
>
> Our old LaVision BioTech and also our new Leica SP8 both support
> sequential scanning with several wavelengths (tuning in-between), I suppose
> the others do too.
>
> I believe the fixed 1046 is useful only if you want to do CARS. Or if you
> happen to have fluors that are nicely excited at that range. But I don't
> know any. In that respect a TiSa-OPO combination may be more useful were
> you can take out 10% of a 4 W laser to image directly (e.g.834 nm) and the
> 90% to generate a long wavelength.
>
> I wouldn't worry too much about cooking. One, the absorption of tissue is
> very low between 1200 and 1300. Two, the transmittance of the microscopes
> is very low, so you might get not so much out of the objective (still
> plenty to do fluorescence). Three, if you are still worried, a resonant
> scanner might be a good idea.
>
> Steffen
>
>
> Am 07.07.2016 um 18:54 schrieb Cammer, Michael:
>
>> *****
>> 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.
>> *****
>>
>> We are considering a multiphoton purchase and a big issue is
>> illumination.  Putting budget constraints aside and thinking purely of
>> imaging non-destructively in live tissues, what would give us the most
>> flexibility for exciting multiple probes for maximum signal while minimally
>> damaging the biology?
>>
>>
>>
>> We are used to using lasers that tune from approx 700 to 1060 nm and
>> mostly use 890 to 930 nm, but this does not provide good red imaging.  If
>> money were no issue, would we be wise to get a laser such as the dual line
>> Insight or two lasers from 690 to 1080 nm?
>>
>>
>>
>> Some of the questions that have come up are:
>>
>> *         We think a laser that tunesup to 1300 nm would solve the red
>> imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of
>> collagen would we need to use a different wavelength?  If so, how long does
>> this take and do the commercial systems support this?
>>
>> *         With a dual line laser that tunes out to 1300 nm is the fixed
>> 1046 nm line really useful?
>>
>> *         Does a dual line system cook the sample?
>>
>>
>>
>> Any thoughts on this (and on specific multiphoton scopes) greatly
>> appreciated.
>>
>>
>>
>> Thank you!
>>
>>
>> =========================================================================
>> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical
>> Center
>> Cell:  914-309-3270     Office: Skirball 2nd Floor main office
>> http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>>
>>
>> ------------------------------------------------------------
>> This email message, including any attachments, is for the sole use of the
>> intended recipient(s) and may contain information that is proprietary,
>> confidential, and exempt from disclosure under applicable law. Any
>> unauthorized review, use, disclosure, or distribution is prohibited. If you
>> have received this email in error please notify the sender by return email
>> and delete the original message. Please note, the recipient should check
>> this email and any attachments for the presence of viruses. The
>> organization accepts no liability for any damage caused by any virus
>> transmitted by this email.
>> =================================
>>
>>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>