Recommendations for commercial multi-photon system purchase

[Adrian Smith-6]

It was my understanding that the 25x is actually smaller (at least in  
back aperture size) than the older 20x 0.95?

The beam delivery path, optics and objective mount on the LaVision  
BioTec are all custom, ie if they don't already cope with the 25x I  
suspect they could easily be made to do. Of course without an  
objective to test who really knows :)

(Furthermore the sample stage is also custom, allowing easy  
accommodation of the extra length)

I would actually like to see a comparison on the LaVision BioTec  
system (or other custom system) of the older 20x Olympus objective  
with the newer 25x objective. It was my understanding (and I may be  
wrong as it was gleaned from interferences from Olympus competitors -  
but supported indirectly by the fine print on at least one Olympus  
objective brochure) that the Olympus systems could not fill the 20x  
objective and hence that objective did not perform as well in laser  
scanning application on a standard Olympus stand as it could have. I  
think (again I may be wrong) that the customisations on the LaVision  
BioTec system largely overcome that limitation. Therefore the  
comparison would be interesting as I wonder how much of the  
improvements shown in the Olympus marketing material are due to better  
filling on the newer objective?

(bit like showing the effect of pre-chirp compensation by using a high  
dispersion system as the base-line - makes the pre-chirp compensation  
look extremely compelling :)

Regards,

Adrian



On 14/05/2009, at 11:17 PM, Watkins, Simon C wrote:

> the objective is considerably larger than the standard oly  
> objectives. both in thread diameter and length so modifications of  
> the stand using oly parts is needed too.
>
> Simon C. Watkins Ph.D, FRCPath
> Professor and Vice Chair, Cell Biology and Physiology
> Professor, Immunology
> Director, Center for Biologic Imaging
> BSTS 225, University of Pittsburgh
> 3500 Terrace St.
> Pittsburgh PA 15261
> Tel: [phone]
> Fax:[phone]
> URL: http://www.cbi.pitt.edu
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Adrian Smith
> Sent: Thursday, May 14, 2009 7:58 AM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
>
> I don't believe there is any technical reason why the 25x Olympus will
> not work on the LaVision BioTec or Prairie systems.
>
> However, my understanding is that Olympus will only sell that
> objective with a new Olympus MPE system... (which makes it a lot more
> than just "super expensive" :).
>
> Extremely disappointing decision on Olympus' part.
>
> I would absolutely love to try the objective on our Olympus microscope
> (supplied by LaVision BioTec) but I am unable to. If it performs as
> well as the marketing material suggests then I would be willing to pay
> a premium for it.
>
> Regards,
>
> Adrian Smith
> Centenary Institute, Sydney, Australia
>
> On 14/05/2009, at 9:23 PM, Watkins, Simon C wrote:
>
>> If and when you try the olympus system make sure they bring you the
>> super expensive dipping/coverslip water optic (25X1.12 NA) which
>> they designed specifically for 2p.  It is absolutely spectacular,
>> and pretty much the only lens we use for all our mpe work nowadays.
>> When you compare systems it may make you lean towards this
>> particular manufacturer.  Of course the LaVision and Prairie scan
>> heads will fit on the Oly stand, not sure whether they will work
>> with this lens but it would be great to find out (anyone out there
>> tried this combo yet).
>> S.
>> Simon C. Watkins Ph.D, FRC Path
>> Professor and Vice Chair Cell Biology and Physiology
>> Professor Immunology
>> Director Center for Biologic Imaging
>> BSTS 225
>> University of Pittsburgh
>> 3500 Terrace St
>> Pittsburgh PA 15261
>> 412-352-2277
>> www.cbi.pitt.edu
>>
>> ________________________________________
>> From: Confocal Microscopy List [[hidden email]] On
>> Behalf Of Adrian Smith [[hidden email]]
>> Sent: Wednesday, May 13, 2009 8:04 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system
>> purchase
>>
>> On 14/05/2009, at 1:57 AM, Alison North wrote:
>>
>>> If you were only interested in multiphoton imaging, the LaVision has
>>> certain advantages, but if you need visible lasers too then I'm
>>> afraid it won't suit your purposes.
>>
>> I'm not sure of the details but the LaVision BioTec did mention some
>> confocal options to me in passing when we were discussing
>> specifications recently. We were not interested so I didn't ask any
>> details but it might be worth clarifying with them what they can
>> currently offer.
>>
>> From recollection I think the Prairie was also MP-only, at least when
>> we were looking (that was one of its selling points). Maybe that has
>> changed or I'm misrembering?
>>
>> Regards,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, Australia

[Adrian Smith-6]

Or have the ability built into the system to adjust for objectives  
from different manufacturers.


On 14/05/2009, at 11:39 PM, Guy Cox wrote:

> Olympus are certainly not the only people to make such a lens.  
> Leica have one (it's almost the size of a beer can!) but that's not  
> much comfort if you are running a third-party system based on an  
> Olympus stand.  I guess there's a caveat emptor here - make sure if  
> you are buying a third-party system on a "Big 4" stand that the  
> microscope manufacturer will warrant to make all new stuff available  
> to you.  Or else buy elsewhere.
>
>                                          Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Cameron, Lisa
> Sent: Thursday, 14 May 2009 11:26 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
>
> Thanks to all for your comments on commercial 2-p systems.
>
> As for the Olympus objective - the rep from the local distributor  
> (which no longer is, since Olympus just went direct) said, Olympus  
> has restricted sale of this "super objective" to people who have or  
> purchase Olympus MPE systems only because they are (or do not want  
> to) lose to the competition. Also something about having to  
> manufacture these lens and not having them available for their own  
> Olympus customers. Seems like if they just stepped up production,  
> they could make money selling them to whomever...
>
> Zeiss as put some effort into making a lens to compete with this -  
> they have a 20x 1.0 WD=1.8 that they say is competing well. I have  
> not compared these two objectives directly myself to be able to make  
> any conclusions yet.
>
> - Lisa
>
> ---------------------------------------
> Lisa Cameron, Ph.D.
> Director of Confocal and Light Microscopy Dana Farber Cancer Institute
> 44 Binney St.; JF 215
> Boston, MA 02115
> Office phone: [phone]
> Fax: [phone]
> Office location: Jimmy Fund Bldg. 220B
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Watkins, Simon C
> Sent: Thursday, May 14, 2009 9:17 AM
> To: [hidden email]
> Subject: Re: [CONFOCALMICROSCOPY] Recommendations for commercial  
> multi-photon system purchase
>
> the objective is considerably larger than the standard oly  
> objectives. both in thread diameter and length so modifications of  
> the stand using oly parts is needed too.
>
> Simon C. Watkins Ph.D, FRCPath
> Professor and Vice Chair, Cell Biology and Physiology Professor,  
> Immunology Director, Center for Biologic Imaging BSTS 225,  
> University of Pittsburgh 3500 Terrace St.
> Pittsburgh PA 15261
> Tel: [phone]
> Fax:[phone]
> URL: http://www.cbi.pitt.edu
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Adrian Smith
> Sent: Thursday, May 14, 2009 7:58 AM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
>
> I don't believe there is any technical reason why the 25x Olympus  
> will not work on the LaVision BioTec or Prairie systems.
>
> However, my understanding is that Olympus will only sell that  
> objective with a new Olympus MPE system... (which makes it a lot  
> more than just "super expensive" :).
>
> Extremely disappointing decision on Olympus' part.
>
> I would absolutely love to try the objective on our Olympus  
> microscope (supplied by LaVision BioTec) but I am unable to. If it  
> performs as well as the marketing material suggests then I would be  
> willing to pay a premium for it.
>
> Regards,
>
> Adrian Smith
> Centenary Institute, Sydney, Australia
>
> On 14/05/2009, at 9:23 PM, Watkins, Simon C wrote:
>
>> If and when you try the olympus system make sure they bring you the
>> super expensive dipping/coverslip water optic (25X1.12 NA) which they
>> designed specifically for 2p.  It is absolutely spectacular,
>> and pretty much the only lens we use for all our mpe work nowadays.
>> When you compare systems it may make you lean towards this particular
>> manufacturer.  Of course the LaVision and Prairie scan heads will fit
>> on the Oly stand, not sure whether they will work with this lens but
>> it would be great to find out (anyone out there tried this combo  
>> yet).
>> S.
>> Simon C. Watkins Ph.D, FRC Path
>> Professor and Vice Chair Cell Biology and Physiology Professor
>> Immunology Director Center for Biologic Imaging BSTS 225 University  
>> of
>> Pittsburgh 3500 Terrace St Pittsburgh PA 15261
>> 412-352-2277
>> www.cbi.pitt.edu
>>
>> ________________________________________
>> From: Confocal Microscopy List [[hidden email]] On
>> Behalf Of Adrian Smith [[hidden email]]
>> Sent: Wednesday, May 13, 2009 8:04 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system
>> purchase
>>
>> On 14/05/2009, at 1:57 AM, Alison North wrote:
>>
>>> If you were only interested in multiphoton imaging, the LaVision has
>>> certain advantages, but if you need visible lasers too then I'm
>>> afraid it won't suit your purposes.
>>
>> I'm not sure of the details but the LaVision BioTec did mention some
>> confocal options to me in passing when we were discussing
>> specifications recently. We were not interested so I didn't ask any
>> details but it might be worth clarifying with them what they can
>> currently offer.
>>
>> From recollection I think the Prairie was also MP-only, at least when
>> we were looking (that was one of its selling points). Maybe that has
>> changed or I'm misrembering?
>>
>> Regards,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, Australia
>
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[Haberman, Ann]

I also have a LaVision which has a removable adaptor for objectives
with larger back apertures. Like Adrian, I am quite happy with our
upright which is dedicated to multiphoton excitation.

We use the Olympus 20X NA 0.95, which is also a lovely lens with a
large back aperture. I have also been told by Olympus reps that they
can't sell the new 25X objective to me unless I purchase the entire
system.

I would like to remind the list that it is possible to compare the
Zeiss 710 and LaVision microscopes at the Yale Microscopy Workshop
June 9-11th. They will both be outfitted with Coherent lasers and
available for use. This annual event provides open access to new
instrumentation and only requires your free registration online.
http://microscopy.med.yale.edu

Ann Haberman
--

Ann Haberman, PhD
Department of Laboratory Medicine
Yale University School of Medicine
1 Gilbert  St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

[Adrian Smith-6]

My information came directly from an Olympus Australia representative.

Regards,

Adrian

On 14/05/2009, at 11:05 PM, Guy Cox wrote:

> This should not be any problem on (for example) a Prairie or  
> LaVision system fitted on an Olympus stand.  I really find it hard  
> to believe that Olympus would not supply this lens for such use.  
> Can anyone actually confirm that they will not?  Is there anyone  
> from Olympus on this list?
>
>                                     Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net <http://www.guycox.net/>
>
>
>
> ________________________________
>
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of RICHARD BURRY
> Sent: Thursday, 14 May 2009 10:47 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
>
>
> The Olympus 25x 1.05 N.A. objective has an enlarged aperture in the  
> back image plane to allow more light throughput.  This requires  
> additional lenses capable of filling the aperture, which Olympus  
> has.  While the lens may fit on other microscopes, with ut the  
> filling of this aperture, it will not perform as well as it could.
>
> Dick
>
> ----- Original Message -----
> From: Adrian Smith <[hidden email]>
> Date: Thursday, May 14, 2009 8:02 am
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
> To: [hidden email]
>
>> I don't believe there is any technical reason why the 25x
>> Olympus will
>> not work on the LaVision BioTec or Prairie systems.
>>
>> However, my understanding is that Olympus will only sell
>> that
>> objective with a new Olympus MPE system... (which makes it a lot
>> more
>> than just "super expensive" :).
>>
>> Extremely disappointing decision on Olympus' part.
>>
>> I would absolutely love to try the objective on our Olympus
>> microscope
>> (supplied by LaVision BioTec) but I am unable to. If it performs
>> as
>> well as the marketing material suggests then I would be willing
>> to pay
>> a premium for it.
>>
>> Regards,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, Australia
>>
>> On 14/05/2009, at 9:23 PM, Watkins, Simon C wrote:
>>
>>> If and when you try the olympus system make sure they bring
>> you the
>>> super expensive dipping/coverslip water optic (25X1.12 NA)
>> which
>>> they designed specifically for 2p.  It is absolutely
>> spectacular,
>>> and pretty much the only lens we use for all our mpe work
>> nowadays.
>>> When you compare systems it may make you lean towards
>> this
>>> particular manufacturer.  Of course the LaVision and
>> Prairie scan
>>> heads will fit on the Oly stand, not sure whether they will
>> work
>>> with this lens but it would be great to find out (anyone out
>> there
>>> tried this combo yet).
>>> S.
>>> Simon C. Watkins Ph.D, FRC Path
>>> Professor and Vice Chair Cell Biology and Physiology
>>> Professor Immunology
>>> Director Center for Biologic Imaging
>>> BSTS 225
>>> University of Pittsburgh
>>> 3500 Terrace St
>>> Pittsburgh PA 15261
>>> 412-352-2277
>>> www.cbi.pitt.edu
>>>
>>> ________________________________________
>>> From: Confocal Microscopy List
>> [[hidden email]] On
>>> Behalf Of Adrian Smith [[hidden email]]
>>> Sent: Wednesday, May 13, 2009 8:04 PM
>>> To: [hidden email]
>>> Subject: Re: Recommendations for commercial multi-photon
>> system
>>> purchase
>>>
>>> On 14/05/2009, at 1:57 AM, Alison North wrote:
>>>
>>>> If you were only interested in multiphoton imaging, the
>> LaVision has
>>>> certain advantages, but if you need visible lasers too then I'm
>>>> afraid it won't suit your purposes.
>>>
>>> I'm not sure of the details but the LaVision BioTec did
>> mention some
>>> confocal options to me in passing when we were discussing
>>> specifications recently. We were not interested so I didn't
>> ask any
>>> details but it might be worth clarifying with them what they can
>>> currently offer.
>>>
>>> From recollection I think the Prairie was also MP-only, at
>> least when
>>> we were looking (that was one of its selling points). Maybe
>> that has
>>> changed or I'm misrembering?
>>>
>>> Regards,
>>>
>>> Adrian Smith
>>> Centenary Institute, Sydney, Australia
>>
>>
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>
> Richard W. Burry, Ph.D.
> Department of Neuroscience, College of Medicine
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> 277 Biomedical Research Tower
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[Beat Ludin]

Hi Sylvie

We're also offering our BOX incubators in black
or any combination of transparent and black (commercial interest, of course).

Best regards,

Beat

At 10:22 14-05-2009, you wrote:

>I think Olympus has now implemented the tuning of the laser and the
>compensation within their software.
>
>Cameron, where did you get these black incubators? I am interested in
>getting one done as well.
>
>Med vänlig hälsning / Best regards
>
>Sylvie
>
>@@@@@@@@@@@@@@@@@@@@@@@@
>Sylvie Le Guyader
>Dept of Biosciences and Nutrition
>Karolinska Institutet
>Novum
>14157 Huddinge
>Sweden
>[phone]
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]] On Behalf Of Cameron
> > Nowell
> > Sent: 14 May 2009 00:28
> > To: [hidden email]
> > Subject: Re: Recommendations for commercial multi-photon system purchase
> >
> > Hi Lisa,
> >
> > Thought i woudl chip in with my experience as well. We have 2 Olympus MPE
> > Systems, one inverted and one upright that share a common Mai Tai Laser
>(the
> > laser can only be sent to one scope at a time). The upright is used
>routinely for
> > intravital imaging and has performed flawlessly. We have imaged zebra fish
>and
> > mice (both live and dead) on it and acheived some spectacular data sets.
> >
> > The current configuration has on large 0.95NA water imersion 20x objective
>on it,
> > but it is easy enough to slip another turret of objectives onto it if
>required. Not sure
> > if it is true for other system but the olympus system is tuned to work
>with the 20x
> > objective in multiphoton mode, so putting other objectives on may not
>result in
> > optimal imaging. For single photon excitation it doesn't matter.
> >
> > The software is stable and easy enough to use. Users are usually up and
>going
> > independently after a coupel of hours of training and practice.
> >
> > The only issue i have with it is that you can not communicate directly
>with the laser
> > via the Olympus software. You can control the laser power via an ND filter
>system
> > but other functions (such as wave length tuning) are carried out using a
>seperate
> > software package installed on a laptop plugged into the laser.
> >
> > One last thing worth mentioning is that both microscoes are fitted with
>black acrylic
> > incubators (thanks to Steve Cody for this). They work so well at blocking
>out light
> > that you can perform MP imaging with the room lights on!
> >
> > Also look into what the service provided by the companies are in your
>area. Here in
> > Melbourne Olympus are probaly one of the best for response and quality of
>service.
> > There is no point getting the best microsocpe out there if the peopel in
>your area
> > are not that good at keeping it going.
> >
> >
> > Cheers
> >
> >
> > Cam
> >
> >
> >
> > Cameron J. Nowell
> > Microscpy Manager
> > Central Resource for Advanced Microscopy
> > Ludwig Insttue for Cancer Research
> > PO Box 2008
> > Royal Melbourne Hospital
> > Victoria, 3050
> > AUSTRALIA
> >
> > Office: [phone]
> > Mobile: [phone]
> > Fax: [phone]
> >
> > http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
> >
> >
> > ________________________________
> >
> > From: Confocal Microscopy List on behalf of Cameron, Lisa
> > Sent: Thu 14/05/2009 12:04 AM
> > To: [hidden email]
> > Subject: Recommendations for commercial multi-photon system purchase
> >
> >
> >
> > I have been investigating commercial multi-photon systems for awhile in
>order to
> > purchase a system for my Institute's core microscopy facility. Our
>interest is to have
> > the capability to do intravital imaging on an upright stand, but also be
>able to have
> > facility users be able to put slides on and use the visible scanner and
>detectors. I
> > realize this is a tall order for such versatility in one system, but since
>it is for a core
> > (which needs to bring in revenue), I'm looking for the most flexible
>system. Does
> > anyone have any suggestions about the most recent systems on the market?
>Or
> > could you point out factors you think are the most important for making
>the decision
> > on which company to go with? Please feel free to contact me off line.
> >
> > I have seen a demo of the Leica SP5 MP, Zeiss 710 NLO, Olympus MPE and
> > Prairie's system.
> >
> > (BTW - my own experience is with widefield and confocal live-cell imaging,
>so I
> > have not done 2-p myself, but have been learning about it for about a
>year)
> >
> > Thanks!
> > - Lisa
> >
> > ---------------------------------------
> > Lisa Cameron, Ph.D.
> > Director of Confocal and Light Microscopy
> > Dana Farber Cancer Institute
> > 44 Binney St.; JF 215
> > Boston, MA 02115
> > Office phone: [phone]
> > Fax: [phone]
> > [hidden email]
> >
> > The information in this e-mail is intended only for the person to whom it
>is
> > addressed. If you believe this e-mail was sent to you in error and the
>e-mail
> > contains patient information, please contact the Partners Compliance
>HelpLine at
> > http://www.partners.org/complianceline . If the e-mail was sent to you in
>error
> > but does not contain patient information, please contact the sender and
>properly
> > dispose of the e-mail.

[Guy Cox]

Well, what can I say?  You recently posted that you are in the market for a second system.  You can just insist that your third-party system must be based on 'brand N' or 'brand L' or even 'brand Z' if 'brand O' will not give you the assurance of optics supply that you need.  The same applies to anyone buying a 3rd-party system.  The marketplace isn't that big - a handful of serious orders carries rather substantial weight.

                                                Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Adrian Smith
Sent: Thursday, 14 May 2009 11:51 PM
To: [hidden email]
Subject: Re: Recommendations for commercial multi-photon system purchase

My information came directly from an Olympus Australia representative.

Regards,

Adrian

On 14/05/2009, at 11:05 PM, Guy Cox wrote:

> This should not be any problem on (for example) a Prairie or LaVision
> system fitted on an Olympus stand.  I really find it hard
> to believe that Olympus would not supply this lens for such use.  
> Can anyone actually confirm that they will not?  Is there anyone from
> Olympus on this list?
>
>                                     Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit,
> Madsen Building F09, University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net <http://www.guycox.net/>
>
>
>
> ________________________________
>
> From: Confocal Microscopy List
> [mailto:[hidden email]
> ] On Behalf Of RICHARD BURRY
> Sent: Thursday, 14 May 2009 10:47 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system
> purchase
>
>
> The Olympus 25x 1.05 N.A. objective has an enlarged aperture in the
> back image plane to allow more light throughput.  This requires
> additional lenses capable of filling the aperture, which Olympus has.  
> While the lens may fit on other microscopes, with ut the filling of
> this aperture, it will not perform as well as it could.
>
> Dick
>
> ----- Original Message -----
> From: Adrian Smith <[hidden email]>
> Date: Thursday, May 14, 2009 8:02 am
> Subject: Re: Recommendations for commercial multi-photon system
> purchase
> To: [hidden email]
>
>> I don't believe there is any technical reason why the 25x Olympus
>> will not work on the LaVision BioTec or Prairie systems.
>>
>> However, my understanding is that Olympus will only sell that
>> objective with a new Olympus MPE system... (which makes it a lot more
>> than just "super expensive" :).
>>
>> Extremely disappointing decision on Olympus' part.
>>
>> I would absolutely love to try the objective on our Olympus
>> microscope (supplied by LaVision BioTec) but I am unable to. If it
>> performs as well as the marketing material suggests then I would be
>> willing to pay a premium for it.
>>
>> Regards,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, Australia
>>
>> On 14/05/2009, at 9:23 PM, Watkins, Simon C wrote:
>>
>>> If and when you try the olympus system make sure they bring
>> you the
>>> super expensive dipping/coverslip water optic (25X1.12 NA)
>> which
>>> they designed specifically for 2p.  It is absolutely
>> spectacular,
>>> and pretty much the only lens we use for all our mpe work
>> nowadays.
>>> When you compare systems it may make you lean towards
>> this
>>> particular manufacturer.  Of course the LaVision and
>> Prairie scan
>>> heads will fit on the Oly stand, not sure whether they will
>> work
>>> with this lens but it would be great to find out (anyone out
>> there
>>> tried this combo yet).
>>> S.
>>> Simon C. Watkins Ph.D, FRC Path
>>> Professor and Vice Chair Cell Biology and Physiology Professor
>>> Immunology Director Center for Biologic Imaging BSTS 225 University
>>> of Pittsburgh 3500 Terrace St Pittsburgh PA 15261
>>> 412-352-2277
>>> www.cbi.pitt.edu
>>>
>>> ________________________________________
>>> From: Confocal Microscopy List
>> [[hidden email]] On
>>> Behalf Of Adrian Smith [[hidden email]]
>>> Sent: Wednesday, May 13, 2009 8:04 PM
>>> To: [hidden email]
>>> Subject: Re: Recommendations for commercial multi-photon
>> system
>>> purchase
>>>
>>> On 14/05/2009, at 1:57 AM, Alison North wrote:
>>>
>>>> If you were only interested in multiphoton imaging, the
>> LaVision has
>>>> certain advantages, but if you need visible lasers too then I'm
>>>> afraid it won't suit your purposes.
>>>
>>> I'm not sure of the details but the LaVision BioTec did
>> mention some
>>> confocal options to me in passing when we were discussing
>>> specifications recently. We were not interested so I didn't
>> ask any
>>> details but it might be worth clarifying with them what they can
>>> currently offer.
>>>
>>> From recollection I think the Prairie was also MP-only, at
>> least when
>>> we were looking (that was one of its selling points). Maybe
>> that has
>>> changed or I'm misrembering?
>>>
>>> Regards,
>>>
>>> Adrian Smith
>>> Centenary Institute, Sydney, Australia
>>
>>
>> --
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>
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> Department of Neuroscience, College of Medicine Campus Microscopy and
> Imaging Facility, Director The Ohio State University Associate Editor,
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[Adrian Smith-6]

Yes - brand "N" is getting a much larger look in from Brand "L" than  
in the past.


On 15/05/2009, at 12:15 AM, Guy Cox wrote:

> Well, what can I say?  You recently posted that you are in the  
> market for a second system.  You can just insist that your third-
> party system must be based on 'brand N' or 'brand L' or even 'brand  
> Z' if 'brand O' will not give you the assurance of optics supply  
> that you need.  The same applies to anyone buying a 3rd-party  
> system.  The marketplace isn't that big - a handful of serious  
> orders carries rather substantial weight.
>
> Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>    http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>     http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ] On Behalf Of Adrian Smith
> Sent: Thursday, 14 May 2009 11:51 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system  
> purchase
>
> My information came directly from an Olympus Australia representative.
>
> Regards,
>
> Adrian
>
> On 14/05/2009, at 11:05 PM, Guy Cox wrote:
>
>> This should not be any problem on (for example) a Prairie or LaVision
>> system fitted on an Olympus stand.  I really find it hard
>> to believe that Olympus would not supply this lens for such use.
>> Can anyone actually confirm that they will not?  Is there anyone from
>> Olympus on this list?
>>
>>                                    Guy
>>
>>
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>   http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope  
>> Unit,
>> Madsen Building F09, University of Sydney, NSW 2006
>> ______________________________________________
>> Phone [phone]     Fax [phone]
>> Mobile [phone]
>> ______________________________________________
>>    http://www.guycox.net <http://www.guycox.net/>
>>
>>
>>
>> ________________________________
>>
>> From: Confocal Microscopy List
>> [mailto:[hidden email]
>> ] On Behalf Of RICHARD BURRY
>> Sent: Thursday, 14 May 2009 10:47 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system
>> purchase
>>
>>
>> The Olympus 25x 1.05 N.A. objective has an enlarged aperture in the
>> back image plane to allow more light throughput.  This requires
>> additional lenses capable of filling the aperture, which Olympus has.
>> While the lens may fit on other microscopes, with ut the filling of
>> this aperture, it will not perform as well as it could.
>>
>> Dick
>>
>> ----- Original Message -----
>> From: Adrian Smith <[hidden email]>
>> Date: Thursday, May 14, 2009 8:02 am
>> Subject: Re: Recommendations for commercial multi-photon system
>> purchase
>> To: [hidden email]
>>
>>> I don't believe there is any technical reason why the 25x Olympus
>>> will not work on the LaVision BioTec or Prairie systems.
>>>
>>> However, my understanding is that Olympus will only sell that
>>> objective with a new Olympus MPE system... (which makes it a lot  
>>> more
>>> than just "super expensive" :).
>>>
>>> Extremely disappointing decision on Olympus' part.
>>>
>>> I would absolutely love to try the objective on our Olympus
>>> microscope (supplied by LaVision BioTec) but I am unable to. If it
>>> performs as well as the marketing material suggests then I would be
>>> willing to pay a premium for it.
>>>
>>> Regards,
>>>
>>> Adrian Smith
>>> Centenary Institute, Sydney, Australia
>>>
>>> On 14/05/2009, at 9:23 PM, Watkins, Simon C wrote:
>>>
>>>> If and when you try the olympus system make sure they bring
>>> you the
>>>> super expensive dipping/coverslip water optic (25X1.12 NA)
>>> which
>>>> they designed specifically for 2p.  It is absolutely
>>> spectacular,
>>>> and pretty much the only lens we use for all our mpe work
>>> nowadays.
>>>> When you compare systems it may make you lean towards
>>> this
>>>> particular manufacturer.  Of course the LaVision and
>>> Prairie scan
>>>> heads will fit on the Oly stand, not sure whether they will
>>> work
>>>> with this lens but it would be great to find out (anyone out
>>> there
>>>> tried this combo yet).
>>>> S.
>>>> Simon C. Watkins Ph.D, FRC Path
>>>> Professor and Vice Chair Cell Biology and Physiology Professor
>>>> Immunology Director Center for Biologic Imaging BSTS 225 University
>>>> of Pittsburgh 3500 Terrace St Pittsburgh PA 15261
>>>> 412-352-2277
>>>> www.cbi.pitt.edu
>>>>
>>>> ________________________________________
>>>> From: Confocal Microscopy List
>>> [[hidden email]] On
>>>> Behalf Of Adrian Smith [[hidden email]]
>>>> Sent: Wednesday, May 13, 2009 8:04 PM
>>>> To: [hidden email]
>>>> Subject: Re: Recommendations for commercial multi-photon
>>> system
>>>> purchase
>>>>
>>>> On 14/05/2009, at 1:57 AM, Alison North wrote:
>>>>
>>>>> If you were only interested in multiphoton imaging, the
>>> LaVision has
>>>>> certain advantages, but if you need visible lasers too then I'm
>>>>> afraid it won't suit your purposes.
>>>>
>>>> I'm not sure of the details but the LaVision BioTec did
>>> mention some
>>>> confocal options to me in passing when we were discussing
>>>> specifications recently. We were not interested so I didn't
>>> ask any
>>>> details but it might be worth clarifying with them what they can
>>>> currently offer.
>>>>
>>>> From recollection I think the Prairie was also MP-only, at
>>> least when
>>>> we were looking (that was one of its selling points). Maybe
>>> that has
>>>> changed or I'm misrembering?
>>>>
>>>> Regards,
>>>>
>>>> Adrian Smith
>>>> Centenary Institute, Sydney, Australia
>>>
>>>
>>> --
>>> BEGIN-ANTISPAM-VOTING-LINKS
>>> ------------------------------------------------------
>>>
>>> Teach CanIt if this mail (ID 868940829) is spam:
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>>> spam:    https://antispam.osu.edu/b.php?
>>> c=n&i=868940829&m=73ec3abfd3ab
>>> Forget vote:
>>> https://antispam.osu.edu/b.php?c=f&i=868940829&m=73ec3abfd3ab----
>>> --------------------------------------------------
>>> END-ANTISPAM-VOTING-LINKS
>>>
>>
>> Richard W. Burry, Ph.D.
>> Department of Neuroscience, College of Medicine Campus Microscopy and
>> Imaging Facility, Director The Ohio State University Associate  
>> Editor,
>> Journal of Histochemistry and Cytochemistry
>> 277 Biomedical Research Tower
>> 460 West Twelfth Avenue
>> Columbus, Ohio 43210
>> Voice 614.292.2814  Cell 614.638.3345  Fax [phone]
>>
>>
>>
>>
>> Internal Virus Database is out-of-date.
>> Checked by AVG.
>> Version: 7.5.557 / Virus Database: 270.12.11/2089 - Release Date:
>> 30/04/2009 5:53 PM
>>
>>
>>
>> Internal Virus Database is out-of-date.
>> Checked by AVG.
>> Version: 7.5.557 / Virus Database: 270.12.11/2089 - Release Date:
>> 30/04/2009 5:53 PM
>>
>
> Internal Virus Database is out-of-date.
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[Stephen Cody-2]

Dear List,

 

Sorry for the belated reply on this thread.

 

Just a couple of minor clarifications. The multiphoton system that Cameron is referring to below was a system that I ordered when I worked at that Institute and Cameron has now inherited. Just to clarify why we both are making comment on this system.

 

As we were making the purchase decision on the brand of multiphoton and IR laser, I received notice that the DeepSee was soon to be released. This happened to coincide with me visiting Olympus R&D in Tokyo. I had permission from SpectraPhysics to discuss with Olympus the immanent release of their new DeepSee laser. We ended up ordering the Olympus system with the DeepSee laser. This was the first purchase of this laser on a multiphoton system. Being the first of it's kind it was understandable that the software did not communicate with the pre-chirp mechanism. Although at the time Zeiss and I think Leica could control the wavelength settings from within their software. It seemed that Olympus would release this feature given time, and if reports on this list are correct it appears that they may have done so now.

 

It is true that the upright microscope multiphoton system at the Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping lens. This was done at installation by fitting and testing with a custom set of lenses for the lens of our choice. I just want to clarify that Olympus could always optimise their system for any objective at the time of installation. I think from memory I chose the 60x WI with cover slip correction for the inverted.

 

I was aware during the entire purchase and installation process that Olympus were developing a motorised beam expander for their MPE systems. I believe that the Olympus motorised beam expander is now available and so you should be able to optimise a new system for any objective with a simple user adjustment.

 

An aside:

Many years ago whilst running a Bio-Rad UV confocal system, we were faced with a similar issue. Bio-Rad in their wisdom would set up their UV beam expander (they called it a "zoom lens" and after I complained renamed it a collimator) with one objective in mind.

I found I always had to adjust this lens (which was frowned upon by Bio-Rad) depending on the specimen or lens chosen. I asked Allan Anderson (then Bio-Rad now ClearState Solutions, the manufacturer of the black incubators Cameron was referring to), if he could make the collimator adjustable without having to open up the UV optics. He bought a small DC electric motor from a hobby shop, and from then on adjusting the beam to suit each lens and specimen was a simple matter. Bio-Rad continued with this strategy in the design of their multiphoton systems. A motorised beam expander, or a series of lenses in a filter wheel, preferably computer controlled is most important if you wish to use a number of different objectives at the extremes of the light spectrum UV or IR.

 

Cheers

Stephen Cody

 

2009/5/14 Cameron Nowell <[hidden email]>

Hi Lisa,

Thought i woudl chip in with my experience as well. We have 2 Olympus MPE Systems, one inverted and one upright that share a common Mai Tai Laser (the laser can only be sent to one scope at a time). The upright is used routinely for intravital imaging and has performed flawlessly. We have imaged zebra fish and mice (both live and dead) on it and acheived some spectacular data sets.

The current configuration has on large 0.95NA water imersion 20x objective on it, but it is easy enough to slip another turret of objectives onto it if required. Not sure if it is true for other system but the olympus system is tuned to work with the 20x objective in multiphoton mode, so putting other objectives on may not result in optimal imaging. For single photon excitation it doesn't matter.

The software is stable and easy enough to use. Users are usually up and going independently after a coupel of hours of training and practice.

The only issue i have with it is that you can not communicate directly with the laser via the Olympus software. You can control the laser power via an ND filter system but other functions (such as wave length tuning) are carried out using a seperate software package installed on a laptop plugged into the laser.

One last thing worth mentioning is that both microscoes are fitted with black acrylic incubators (thanks to Steve Cody for this). They work so well at blocking out light that you can perform MP imaging with the room lights on!

Also look into what the service provided by the companies are in your area. Here in Melbourne Olympus are probaly one of the best for response and quality of service. There is no point getting the best microsocpe out there if the peopel in your area are not that good at keeping it going.


Cheers


Cam



Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm


________________________________

From: Confocal Microscopy List on behalf of Cameron, Lisa
Sent: Thu 14/05/2009 12:04 AM

To: [hidden email]
Subject: Recommendations for commercial multi-photon system purchase

I have been investigating commercial multi-photon systems for awhile in order to purchase a system for my Institute's core microscopy facility. Our interest is to have the capability to do intravital imaging on an upright stand, but also be able to have facility users be able to put slides on and use the visible scanner and detectors. I realize this is a tall order for such versatility in one system, but since it is for a core (which needs to bring in revenue), I'm looking for the most flexible system. Does anyone have any suggestions about the most recent systems on the market? Or could you point out factors you think are the most important for making the decision on which company to go with? Please feel free to contact me off line.

I have seen a demo of the Leica SP5 MP, Zeiss 710 NLO, Olympus MPE and Prairie's system.

(BTW - my own experience is with widefield and confocal live-cell imaging, so I have not done 2-p myself, but have been learning about it for about a year)

Thanks!
- Lisa

---------------------------------------
Lisa Cameron, Ph.D.
Director of Confocal and Light Microscopy
Dana Farber Cancer Institute
44 Binney St.; JF 215
Boston, MA 02115
Office phone: [phone]
Fax: [phone]
[hidden email]

The information in this e-mail is intended only for the person to whom it is
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[Adrian Smith-6]

On 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> It is true that the upright microscope multiphoton system at the  
> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping  
> lens. This was done at installation by fitting and testing with a  
> custom set of lenses for the lens of our choice. I just want to  
> clarify that Olympus could always optimise their system for any  
> objective at the time of installation. I think from memory I chose  
> the 60x WI with cover slip correction for the inverted.


So, the question still stands - has anyone directly tested the older  
20x dipping lens (with tuning to fill the back aperture) against the  
new 25x? (and is allowed to share the data?)

Apparently a theoretical question for those of with existing systems  
but of interest none the less :)

Steve (or Cameron) - you might also be able to comment of the  
performance of the Olympus system with the pre-chirp on the DeepSee on/
off (assuming you can turn the compensation off?)


Regards,
Adrian Smith, Centenary Institute

[Cameron Nowell]

Hi Adrian,
 
After just having a brief look I don't think the pre-chirp unit can be disabled, there is no off switch and I am pretty sure if I disconnect the control box all sorts of weird things will go wrong. There is already one redundant component on the Maintain system that can not be removed for that reason.
 
I can say that if it is not set optimally there is a reduction in image quality and penetration depth. Although it does seem very dependent on the sample and the fluorophore. On some samples you can gain an extra 100 or more micron in the depth by tuning the pre-chirp. Other samples not a lot seems to happen.
 
I have spoken to Olympus about getting the 25x lens but unfortunately there are two problems. 1. it is scarily expensive 2. our system would have to be changed a bit to accommodate it (more expense). I would be curious to see any comparisons if anyone has them, maybe it could be worth the expense:) That being said though I am very, very happy with the quality we are getting from the 20x lens.
 
 
Cheers
 
 
Cam
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

________________________________

From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Fri 29/05/2009 5:25 AM
To: [hidden email]
Subject: Re: Recommendations for commercial multi-photon system purchase



On 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> It is true that the upright microscope multiphoton system at the
> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
> lens. This was done at installation by fitting and testing with a
> custom set of lenses for the lens of our choice. I just want to
> clarify that Olympus could always optimise their system for any
> objective at the time of installation. I think from memory I chose
> the 60x WI with cover slip correction for the inverted.


So, the question still stands - has anyone directly tested the older
20x dipping lens (with tuning to fill the back aperture) against the
new 25x? (and is allowed to share the data?)

Apparently a theoretical question for those of with existing systems
but of interest none the less :)

Steve (or Cameron) - you might also be able to comment of the
performance of the Olympus system with the pre-chirp on the DeepSee on/
off (assuming you can turn the compensation off?)


Regards,
Adrian Smith, Centenary Institute

[Andreas Bruckbauer]

We got brighter images at same laser power settings with the 25x which are less fuzzy in depth, maybe an extra 30 micron in depth in our lymphnodes compared to the 20x. Not a massive effect but clearly visible. Exact comparissons are a bit tricky what would you like to compare? The same laserpower out of the objective for both? At which wavelength? The back aperture of the 20x is a bit underfilled so you get more power through using same optics. Now that we have the motorized Kepler optics working i might run some more tests.

best wishes

Andreas

-----Original Message-----
From: Cameron Nowell <[hidden email]>
To: [hidden email]
Sent: Fri, 29 May 2009 0:02
Subject: Re: Recommendations for commercial multi-photon system purchase

Hi Adrian,

 

After just having a brief look I don't think the pre-chirp unit can be disabled, 

there is no off switch and I am pretty sure if I disconnect the control box all 

sorts of weird things will go wrong. There is already one redundant component on 

the Maintain system that can not be removed for that reason.

 

I can say that if it is not set optimally there is a reduction in image quality 

and penetration depth. Although it does seem very dependent on the sample and 

the fluorophore. On some samples you can gain an extra 100 or more micron in the 

depth by tuning the pre-chirp. Other samples not a lot seems to happen.

 

I have spoken to Olympus about getting the 25x lens but unfortunately there are 

two problems. 1. it is scarily expensive 2. our system would have to be changed 

a bit to accommodate it (more expense). I would be curious to see any 

comparisons if anyone has them, maybe it could be worth the expense:) That being 

said though I am very, very happy with the quality we are getting from the 20x 

lens.

 

 

Cheers

 

 

Cam

 

 

Cameron J. Nowell

Microscpy Manager

Central Resource for Advanced Microscopy

Ludwig Insttue for Cancer Research

PO Box 2008

Royal Melbourne Hospital

Victoria, 3050

AUSTRALIA

 

Office: [phone]

Mobile: [phone]

Fax: [phone]

 

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

 



________________________________



From: Confocal Microscopy List on behalf of Adrian Smith

Sent: Fri 29/05/2009 5:25 AM

To: [hidden email]

Subject: Re: Recommendations for commercial multi-photon system purchase







On 26/05/2009, at 4:15 AM, Stephen Cody wrote:



> It is true that the upright microscope multiphoton system at the 

> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping 

> lens. This was done at installation by fitting and testing with a 

> custom set of lenses for the lens of our choice. I just want to 

> clarify that Olympus could always optimise their system for any 

> objective at the time of installation. I think from memory I chose 

> the 60x WI with cover slip correction for the inverted.





So, the question still stands - has anyone directly tested the older 

20x dipping lens (with tuning to fill the back aperture) against the 

new 25x? (and is allowed to share the data?)



Apparently a theoretical question for those of with existing systems 

but of interest none the less :)



Steve (or Cameron) - you might also be able to comment of the 

performance of the Olympus system with the pre-chirp on the DeepSee on/

off (assuming you can turn the compensation off?)





Regards,

Adrian Smith, Centenary Institute

---

Click here to get the very best of AOL, including news, sport, gossip, lifestyles updates and email.

[Stephen Cody-2]

G'day Adrian,

 

As I understand it, the Olympus special 25x dipping objective for multiphoton was only available in the US when we ordered and installed the Ludwig system (Melbourne, Australia). I tried in vein to get the 25X objective. I think it may now be available in Australia, but even so it would require a refit of the beam expander to install on the Ludwig system. Olympus are very precise when installing, customising and testing of their beam expander. It is definitely the most labour intensive part of the installation. They not only ensure that the beam fills the back focal plane of the chosen objective, they measure the beam intensity profile across the beam at this plane, and make adjustments to the lens set in the beam expander kit to maximise performance. This data is recorded and sent back to Tokyo for approval (well that's what seemed to be happening, I could be mistaken). So sadly such a comparison of lenses is not feasible. If and when someone has the motorised beam expander this should be a trivial test.

 

A similar story with the DeepSee. The pre-chirp optics are always in the path of the laser, there is no pre-chirp bypass. I think this is perhaps a project that would be more suited to an optics lab, rather than an instrument built to investigate biology. Testing the benefit of a pre-chirp would take a system such as this offline for at least a couple of months and would also require many, many hours of labour by the Olympus engineer to reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp would require dismantling the the DeepSee itself and reinstalling the IR laser, and then repeating the installation process again to get back to the original configuration. Unfortunately not a practical thing to test. What I can tell you is that it is a very simple matter to adjust the the pre-chirp with your specimen to obtain the brightest signal. A poorly set pre-chirp compared to an adjusted pre-chirp makes quite a difference. But without knowing what the scenario would be with no pre-chirp it is very difficult to give a precise answer.

 

My gut feeling is pre-chirping is fantastic, although Cameron has certainly used it much more than I, so I deffer to him in this regard. Logically, if you can see a clear benefit of adjusting the pre-chirp (more fluorescence and so laser power can be reduced). One would think that this must be better than a system with no pre-chirp where the pulse is spread ("chirped") by the optics of the microscope, and cannot be compensated. I don't want to get into brand name comparisons but I think it important to to let people know that (as explained to me) It seems that different microscope manufacturers have markedly different amounts of chirp associated with their Optics. So the decision "to Pre-Chirp or not to Pre-Chirp" is far more important depending on the brand of microscope you decide to go with. As this was the first installation of its kind, little was known by anyone at the time what was required. It turned out that originally the DeepSee imparted too much correction for our Olympus scope, and SpectraPhysics very quickly supplied a "Low Dispersion" modification to the setup. After the modification we could find the "sweet spot" with the DeepSee pre-chirp extremely easily.

 

The point being with at least some other brands, pre-chirping is probably much more important. I would ignore the "company speak" that maintains that a similar benefit can be achieved simply by increasing IR laser power. I'm not sure if the Multiphoton List is still going (I think it's membership was merged with this list). However the subject "to Pre-Chirp or not to Pre-Chirp" was raised by myself on that list a couple of years ago. Someone replied to me (I think personally not via the list), that they demonstrated a system: Without pre-chirp they observed damage to their tissue in a whole mouse, above and below the plane of focus, but the fluorophore at the plane of focus was not bleached. This suggested direct IR damage of the laser, but that the 2P effect at the fluorophore was not damaging. When the pre-chirp was installed (or perhaps it was adjusted correctly) this direct IR damage was not seen, presumably lower laser power could be used, to achieve the multiphoton effect. I'm sorry I can't remember who gave me this information and I don't have access to my old email anymore. But a big thank you, that bit of information was exactly what I needed to make a decision and allowed me to cut through the "company speak".

 

I would imagine that a collaboration with SpectraPhysics and a microscope company, so that the pre-chirp could be bypassed would be the best way forward to test this. Does SpectraPhysics and a microscope company want to loan me a multiphoton for 12 months (or 12 years for that matter)? I have not mentioned Coherent, at the time they were only offering a third party pre-chirp option. However, Coherent now offer the "Chameleon Vision" with integrated pre-chirp, this too should be considered in any MPE purchase decision.

 

I have no commercial affiliation with any of the companies mentioned.

 

Cheers

Steve

 

Stephen H. Cody
Imaging Research Fellow & Manager
Monash Micro Imaging – AMREP
Monash University
6 Floor Burnet Tower
Alfred Medical Research & Education Precinct
89 Commercial Rd, Melbourne, Australia, 3004

www.microimaging.monash.org
Phone (Monash):  (613) 990 30142
Phone (BakerIDI): (613) 8532 1580

2009/5/29 Cameron Nowell <[hidden email]>

Hi Adrian,

After just having a brief look I don't think the pre-chirp unit can be disabled, there is no off switch and I am pretty sure if I disconnect the control box all sorts of weird things will go wrong. There is already one redundant component on the Maintain system that can not be removed for that reason.

I can say that if it is not set optimally there is a reduction in image quality and penetration depth. Although it does seem very dependent on the sample and the fluorophore. On some samples you can gain an extra 100 or more micron in the depth by tuning the pre-chirp. Other samples not a lot seems to happen.

I have spoken to Olympus about getting the 25x lens but unfortunately there are two problems. 1. it is scarily expensive 2. our system would have to be changed a bit to accommodate it (more expense). I would be curious to see any comparisons if anyone has them, maybe it could be worth the expense:) That being said though I am very, very happy with the quality we are getting from the 20x lens.

Cheers


Cam


Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm


________________________________

From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Fri 29/05/2009 5:25 AM

To: [hidden email]

Subject: Re: Recommendations for commercial multi-photon system purchase

On 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> It is true that the upright microscope multiphoton system at the
> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
> lens. This was done at installation by fitting and testing with a
> custom set of lenses for the lens of our choice. I just want to
> clarify that Olympus could always optimise their system for any
> objective at the time of installation. I think from memory I chose
> the 60x WI with cover slip correction for the inverted.


So, the question still stands - has anyone directly tested the older
20x dipping lens (with tuning to fill the back aperture) against the
new 25x? (and is allowed to share the data?)

Apparently a theoretical question for those of with existing systems
but of interest none the less :)

Steve (or Cameron) - you might also be able to comment of the
performance of the Olympus system with the pre-chirp on the DeepSee on/
off (assuming you can turn the compensation off?)


Regards,
Adrian Smith, Centenary Institute

--
Stephen H. Cody

[Evangelos Gatzogiannis]

   There were some good comments on chirp and pre-chirping an IR pulse for multiphoton applications.  I don’t have a DeepSee or Chameleon Vision, but have/had older Tsunami oscillators and Coherent Mira oscillators.  In both cases I’ve built my own pre-chirp prism pair assemblies.  There is no choice, in my opinion, between no pre-chirp an chirping.   Some of the objective lenses from several manufacturers such as 20x that have less glass don’t chirp IR pulses as much if you are operating greater than ~100fs.  You must, however, compensate for system optics if you are using 60x or 100x oil objectives and going through thick samples.  I have microscopes and objectives from Zeiss, Olympus, and Leica and there are differences – adjustments to a pre-chirp prism pair must be made daily in my lab to compensate for beam pointing stability, to address the different samples my users have, and to address the different objective lenses and microscopes my users work on.   I like having an open accessible prism pair assembly because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300 confocal and between objectives, scanhead optics, and samples, I find that I have to adjust this assembly frequently to optimize the image on a variety of samples.  Also, if damage to your sample is a serious problem, and if you have significant signal – get a Pockel’s cell or similar modulator.  The 76Mhz repetition rate of commercial laser oscillators is like a machine-gun operating away, sometimes a hundreds of kHz – 1-2 Mhz rep rate can be better.  My regards to all you out there with the Mai Tai Deep See, great laser, you can’t do better.  The marriage between  a  Mai Tai Depp See and an FV1000 is a very good one.

 

Good day,

 

Evangelos Gatzogiannis

Ad

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Cody
Sent: Friday, May 29, 2009 10:49 PM
To: [hidden email]
Subject: Re: Recommendations for commercial multi-photon system purchase

 

G'day Adrian,

 

As I understand it, the Olympus special 25x dipping objective for multiphoton was only available in the US when we ordered and installed the Ludwig system (Melbourne, Australia). I tried in vein to get the 25X objective. I think it may now be available in Australia, but even so it would require a refit of the beam expander to install on the Ludwig system. Olympus are very precise when installing, customising and testing of their beam expander. It is definitely the most labour intensive part of the installation. They not only ensure that the beam fills the back focal plane of the chosen objective, they measure the beam intensity profile across the beam at this plane, and make adjustments to the lens set in the beam expander kit to maximise performance. This data is recorded and sent back to Tokyo for approval (well that's what seemed to be happening, I could be mistaken). So sadly such a comparison of lenses is not feasible. If and when someone has the motorised beam expander this should be a trivial test.

 

A similar story with the DeepSee. The pre-chirp optics are always in the path of the laser, there is no pre-chirp bypass. I think this is perhaps a project that would be more suited to an optics lab, rather than an instrument built to investigate biology. Testing the benefit of a pre-chirp would take a system such as this offline for at least a couple of months and would also require many, many hours of labour by the Olympus engineer to reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp would require dismantling the the DeepSee itself and reinstalling the IR laser, and then repeating the installation process again to get back to the original configuration. Unfortunately not a practical thing to test. What I can tell you is that it is a very simple matter to adjust the the pre-chirp with your specimen to obtain the brightest signal. A poorly set pre-chirp compared to an adjusted pre-chirp makes quite a difference. But without knowing what the scenario would be with no pre-chirp it is very difficult to give a precise answer.

 

My gut feeling is pre-chirping is fantastic, although Cameron has certainly used it much more than I, so I deffer to him in this regard. Logically, if you can see a clear benefit of adjusting the pre-chirp (more fluorescence and so laser power can be reduced). One would think that this must be better than a system with no pre-chirp where the pulse is spread ("chirped") by the optics of the microscope, and cannot be compensated. I don't want to get into brand name comparisons but I think it important to to let people know that (as explained to me) It seems that different microscope manufacturers have markedly different amounts of chirp associated with their Optics. So the decision "to Pre-Chirp or not to Pre-Chirp" is far more important depending on the brand of microscope you decide to go with. As this was the first installation of its kind, little was known by anyone at the time what was required. It turned out that originally the DeepSee imparted too much correction for our Olympus scope, and SpectraPhysics very quickly supplied a "Low Dispersion" modification to the setup. After the modification we could find the "sweet spot" with the DeepSee pre-chirp extremely easily.

 

The point being with at least some other brands, pre-chirping is probably much more important. I would ignore the "company speak" that maintains that a similar benefit can be achieved simply by increasing IR laser power. I'm not sure if the Multiphoton List is still going (I think it's membership was merged with this list). However the subject "to Pre-Chirp or not to Pre-Chirp" was raised by myself on that list a couple of years ago. Someone replied to me (I think personally not via the list), that they demonstrated a system: Without pre-chirp they observed damage to their tissue in a whole mouse, above and below the plane of focus, but the fluorophore at the plane of focus was not bleached. This suggested direct IR damage of the laser, but that the 2P effect at the fluorophore was not damaging. When the pre-chirp was installed (or perhaps it was adjusted correctly) this direct IR damage was not seen, presumably lower laser power could be used, to achieve the multiphoton effect. I'm sorry I can't remember who gave me this information and I don't have access to my old email anymore. But a big thank you, that bit of information was exactly what I needed to make a decision and allowed me to cut through the "company speak".

 

I would imagine that a collaboration with SpectraPhysics and a microscope company, so that the pre-chirp could be bypassed would be the best way forward to test this. Does SpectraPhysics and a microscope company want to loan me a multiphoton for 12 months (or 12 years for that matter)? I have not mentioned Coherent, at the time they were only offering a third party pre-chirp option. However, Coherent now offer the "Chameleon Vision" with integrated pre-chirp, this too should be considered in any MPE purchase decision.

 

I have no commercial affiliation with any of the companies mentioned.

 

Cheers

Steve

 

Stephen H. Cody
Imaging Research Fellow & Manager
Monash Micro Imaging – AMREP
Monash University
6 Floor Burnet Tower
Alfred Medical Research & Education Precinct
89 Commercial Rd, Melbourne, Australia, 3004

www.microimaging.monash.org
Phone (Monash):  (613) 990 30142
Phone (BakerIDI): (613) 8532 1580

 

2009/5/29 Cameron Nowell <[hidden email]>

Hi Adrian,

After just having a brief look I don't think the pre-chirp unit can be disabled, there is no off switch and I am pretty sure if I disconnect the control box all sorts of weird things will go wrong. There is already one redundant component on the Maintain system that can not be removed for that reason.

I can say that if it is not set optimally there is a reduction in image quality and penetration depth. Although it does seem very dependent on the sample and the fluorophore. On some samples you can gain an extra 100 or more micron in the depth by tuning the pre-chirp. Other samples not a lot seems to happen.

I have spoken to Olympus about getting the 25x lens but unfortunately there are two problems. 1. it is scarily expensive 2. our system would have to be changed a bit to accommodate it (more expense). I would be curious to see any comparisons if anyone has them, maybe it could be worth the expense:) That being said though I am very, very happy with the quality we are getting from the 20x lens.

Cheers


Cam


Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm


________________________________

From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Fri 29/05/2009 5:25 AM

To: [hidden email]

Subject: Re: Recommendations for commercial multi-photon system purchase

On 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> It is true that the upright microscope multiphoton system at the
> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
> lens. This was done at installation by fitting and testing with a
> custom set of lenses for the lens of our choice. I just want to
> clarify that Olympus could always optimise their system for any
> objective at the time of installation. I think from memory I chose
> the 60x WI with cover slip correction for the inverted.


So, the question still stands - has anyone directly tested the older
20x dipping lens (with tuning to fill the back aperture) against the
new 25x? (and is allowed to share the data?)

Apparently a theoretical question for those of with existing systems
but of interest none the less :)

Steve (or Cameron) - you might also be able to comment of the
performance of the Olympus system with the pre-chirp on the DeepSee on/
off (assuming you can turn the compensation off?)


Regards,
Adrian Smith, Centenary Institute

--
Stephen H. Cody

[RICHARD BURRY]

Cody

 

Great discussion of the advantages and problems with the negative dispersion / pre-chirp systems.  In my limited experience at a demo, the penetration of the Olympus system in fixed tissue was amazing.  We have new Olympus system that will be installed in June so I will have more information then.  Also an update, Olympus now has a motorized beam expander that can be moved into an out of the beam just above the objective. 

 

Dick

----- Original Message -----
From: Stephen Cody <[hidden email]>
Date: Friday, May 29, 2009 10:49 pm
Subject: Re: Recommendations for commercial multi-photon system purchase
To: [hidden email]

> G'day Adrian,
 
> As I understand it, the Olympus special 25x dipping objective for multiphoton was only available in the US when we ordered and installed the Ludwig system (Melbourne, Australia). I tried in vein to get the 25X objective. I think it may now be available in Australia, but even so it would require a refit of the beam expander to install on the Ludwig system. Olympus are very precise when installing, customising and testing of their beam expander. It is definitely the most labour intensive part of the installation. They not only ensure that the beam fills the back focal plane of the chosen objective, they measure the beam intensity profile across the beam at this plane, and make adjustments to the lens set in the beam expander kit to maximise performance. This data is recorded and sent back to Tokyo for approval (well that's what seemed to be happening, I could be mistaken). So sadly such a comparison of lenses is not feasible. If and when someone has the motorised beam expander this should be a trivial test.
 
> A similar story with the DeepSee. The pre-chirp optics are always in the path of the laser, there is no pre-chirp bypass. I think this is perhaps a project that would be more suited to an optics lab, rather than an instrument built to investigate biology. Testing the benefit of a pre-chirp would take a system such as this offline for at least a couple of months and would also require many, many hours of labour by the Olympus engineer to reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp would require dismantling the the DeepSee itself and reinstalling the IR laser, and then repeating the installation process again to get back to the original configuration. Unfortunately not a practical thing to test. What I can tell you is that it is a very simple matter to adjust the the pre-chirp with your specimen to obtain the brightest signal. A poorly set pre-chirp compared to an adjusted pre-chirp makes quite a difference. But without knowing what the scenario would be with no pre-chirp it is very difficult to give a precise answer.
 
> My gut feeling is pre-chirping is fantastic, although Cameron has certainly used it much more than I, so I deffer to him in this regard. Logically, if you can see a clear benefit of adjusting the pre-chirp (more fluorescence and so laser power can be reduced). One would think that this must be better than a system with no pre-chirp where the pulse is spread ("chirped") by the optics of the microscope, and cannot be compensated. I don't want to get into brand name comparisons but I think it important to to let people know that (as explained to me) It seems that different microscope manufacturers have markedly different amounts of chirp associated with their Optics. So the decision "to Pre-Chirp or not to Pre-Chirp" is far more important depending on the brand of microscope you decide to go with. As this was the first installation of its kind, little was known by anyone at the time what was required. It turned out that originally the DeepSee imparted too much correction for our Olympus scope, and SpectraPhysics very quickly supplied a "Low Dispersion" modification to the setup. After the modification we could find the "sweet spot" with the DeepSee pre-chirp extremely easily.
 
> The point being with at least some other brands, pre-chirping is probably much more important. I would ignore the "company speak" that maintains that a similar benefit can be achieved simply by increasing IR laser power. I'm not sure if the Multiphoton List is still going (I think it's membership was merged with this list). However the subject "to Pre-Chirp or not to Pre-Chirp" was raised by myself on that list a couple of years ago. Someone replied to me (I think personally not via the list), that they demonstrated a system: Without pre-chirp they observed damage to their tissue in a whole mouse, above and below the plane of focus, but the fluorophore at the plane of focus was not bleached. This suggested direct IR damage of the laser, but that the 2P effect at the fluorophore was not damaging. When the pre-chirp was installed (or perhaps it was adjusted correctly) this direct IR damage was not seen, presumably lower laser power could be used, to achieve the multiphoton effect. I'm sorry I can't remember who gave me this information and I don't have access to my old email anymore. But a big thank you, that bit of information was exactly what I needed to make a decision and allowed me to cut through the "company speak".
 
> I would imagine that a collaboration with SpectraPhysics and a microscope company, so that the pre-chirp could be bypassed would be the best way forward to test this. Does SpectraPhysics and a microscope company want to loan me a multiphoton for 12 months (or 12 years for that matter)? I have not mentioned Coherent, at the time they were only offering a third party pre-chirp option. However, Coherent now offer the "Chameleon Vision" with integrated pre-chirp, this too should be considered in any MPE purchase decision.
 
> I have no commercial affiliation with any of the companies mentioned.
 
> Cheers
> Steve
 
> Stephen H. Cody
> Imaging Research Fellow & Manager
> Monash Micro Imaging – AMREP
> Monash University
> 6 Floor Burnet Tower
> Alfred Medical Research & Education Precinct
> 89 Commercial Rd, Melbourne, Australia, 3004

> www.microimaging.monash.org
> Phone (Monash):  (613) 990 30142
> Phone (BakerIDI): (613) 8532 1580

> 2009/5/29 Cameron Nowell <<A href="javascript:main.compose('new','t=[e-mail]')">Cameron.Nowell@...>

> Hi Adrian,

> After just having a brief look I don't think the pre-chirp unit can be disabled, there is no off switch and I am pretty sure if I disconnect the control box all sorts of weird things will go wrong. There is already one redundant component on the Maintain system that can not be removed for that reason.

> I can say that if it is not set optimally there is a reduction in image quality and penetration depth. Although it does seem very dependent on the sample and the fluorophore. On some samples you can gain an extra 100 or more micron in the depth by tuning the pre-chirp. Other samples not a lot seems to happen.

> I have spoken to Olympus about getting the 25x lens but unfortunately there are two problems. 1. it is scarily expensive 2. our system would have to be changed a bit to accommodate it (more expense). I would be curious to see any comparisons if anyone has them, maybe it could be worth the expense:) That being said though I am very, very happy with the quality we are getting from the 20x lens.

> Cheers


> Cam


> Cameron J. Nowell
> Microscpy Manager
> Central Resource for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA

> Office: [phone]
> Mobile: [phone]
> Fax: [phone]

> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm


> ________________________________


> From: Confocal Microscopy List on behalf of Adrian Smith
> Sent: Fri 29/05/2009 5:25 AM

> To: <A href="javascript:main.compose('new','t=[e-mail]')">CONFOCALMICROSCOPY@...

> Subject: Re: Recommendations for commercial multi-photon system purchase

> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> > It is true that the upright microscope multiphoton system at the
> > Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
> > lens. This was done at installation by fitting and testing with a
> > custom set of lenses for the lens of our choice. I just want to
> > clarify that Olympus could always optimise their system for any
> > objective at the time of installation. I think from memory I chose
> > the 60x WI with cover slip correction for the inverted.


> So, the question still stands - has anyone directly tested the older
> 20x dipping lens (with tuning to fill the back aperture) against the
> new 25x? (and is allowed to share the data?)

> Apparently a theoretical question for those of with existing systems
> but of interest none the less :)

> Steve (or Cameron) - you might also be able to comment of the
> performance of the Olympus system with the pre-chirp on the DeepSee on/
> off (assuming you can turn the compensation off?)


> Regards,
> Adrian Smith, Centenary Institute

> --
> Stephen H. Cody

---

[Andreas Bruckbauer]

Hi,
as i understand the pre chirp at the Coherent Chameleon vision II can be tuned from 0 to maximum compensation, so that testing a system with and without pre chirp should be easy compared to the DeepSee. According to Olympus and Zeiss reps both companies have tested the Coherent laser and can fit it to their mics.

best wishes

Andreas

-----Original Message-----
From: Stephen Cody <[hidden email]>
To: [hidden email]
Sent: Sat, 30 May 2009 3:49
Subject: Re: Recommendations for commercial multi-photon system purchase

G'day Adrian,

 

As I understand it, the Olympus special 25x dipping objective for multiphoton was only available in the US when we ordered and installed the Ludwig system (Melbourne, Australia). I tried in vein to get the 25X objective. I think it may now be available in Australia, but even so it would require a refit of the beam expander to install on the Ludwig system. Olympus are very precise when installing, customising and testing of their beam expander. It is definitely the most labour intensive part of the installation. They not only ensure that the beam fills the back focal plane of the chosen objective, they measure the beam intensity profile across the beam at this plane, and make adjustments to the lens set in the20beam expander kit to maximise performance. This data is recorded and sent back to Tokyo for approval (well that's what seemed to be happening, I could be mistaken). So sadly such a comparison of lenses is not feasible. If and when someone has the motorised beam expander this should be a trivial test.

 

A similar story with the DeepSee. The pre-chirp optics are always in the path of the laser, there is no pre-chirp bypass. I think this is perhaps a project that would be more suited to an optics lab, rather than an instrument built to investigate biology. Testing the benefit of a pre-chirp would take a system such as this offline for at least a couple of months and would also require many, many hours of labour by the Olympus engineer to reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp would require dismantling the the DeepSee itself and reinstalling the IR laser, and then repeating the installation process again to get back to the original configuration. Unfortunately not a practical thing to test. What I can tell you is that it is a very simple matter to adjust the the pre-chirp with your specimen to obtain the brightest signal. A poorly set pre-chirp compared to an adjusted pre-chirp makes quite a difference. But without knowing what the scenario would be with no pre-chirp it is very difficult to give a precise answer.

 

My gut feeling is pre-chirping is fantastic, althoug h Cameron has certainly used it much more than I, so I deffer to him in this regard. Logically, if you can see a clear benefit of adjusting the pre-chirp (more fluorescence and so laser power can be reduced). One would think that this must be better than a system with no pre-chirp where the pulse is spread ("chirped") by the optics of the microscope, and cannot be compensated. I don't want to get into brand name comparisons but I think it important to to let people know that (as explained to me) It seems that different microscope manufacturers have markedly different amounts of chirp associated with their Optics. So the decision "to Pre-Chirp or not to Pre-Chirp" is far more important depending on the brand of microscope you decide to go with. As this was the first installation of its kind, little was known by anyone at the time what was required. It turned out that originally the DeepSee imparted too much correction for our Olympus scope, and SpectraPhysics very quickly supplied a "Low Dispersion" modification to the setup. After the modification we could find the "sweet spot" with the DeepSee pre-chirp extremely easily.

 

The point being with at least some other brands, pre-chirping is probably much more important. I would ignore the "company speak" that maintains that a similar benefit can be achieved simply by increasing IR laser power. I'm not sure if the Multiphoton List is still going (I think it's membership was merged with this list). However the subject "to Pre-Chirp or not to Pre-Chirp" was raised by myself on that list a couple of years ago. Someone replied to me (I think personally not via the list), that they demonstrated a system: Without pre-chirp they observed damage to their tissue in a whole mouse, above and below the plane of focus, but the fluorophore at the plane of focus was not bleached. This suggested direct IR damage of the laser, but that the 2P effect at the fluorophore was not damaging. When the pre-chirp was installed (or perhaps it was adjusted correctly) this direct IR damage was not seen, presumably lower laser power could be used, to achieve the multiphoton effect. I'm sorry I can't remember who gave me this information and I don't have access to my old email anymore. But a big thank you, that bit of information was exactly what I needed to make a decision and allowed me to cut through the "company speak".

 

I would imagine that a collaboration with SpectraPhysics and a microscope company, so that the pre-chirp could be bypassed would be the best way forward to test this. Does SpectraPhysics and a microscope company want to loan me a multiphoton for 12 months (or 12 years for that matter)? I have not mentioned Coherent, at the time they were only offering a third party pre-chirp option. However, Coherent now offer the "Chameleon Vision" with integrated pre-chirp, this too should be considered in any MPE purchase decision.

 

I have no commercial affi liation with any of the companies mentioned.

 

Cheers

Steve

 

Stephen H. Cody
Imaging Research Fellow & Manager
Monash Micro Imaging – AMREP
Monash University
6 Floor Burnet Tower
Alfred Medical Research & Education Precinct
89 Commercial Rd, Melbourne, Australia, 3004

www.microimaging.monash.org
Phone (Monash):  (613) 990 30142
Phone (BakerIDI): (613) 8532 1580

2009/5/29 Cameron Nowell <[hidden email]>

Hi Adrian,

After just having a brief look I don't think the pre-chirp unit can be disabled, there is no off switch and I am pretty sure if I disconnect the control box all sorts of weird things will go wrong. There is already one redundant component on the Maintain system that can not be removed for that reason.

I can say that if it is not set optimally there is a reductio n in image quality and penetration depth. Although it does seem very dependent on the sample and the fluorophore. On some samples you can gain an extra 100 or more micron in the depth by tuning the pre-chirp. Other samples not a lot seems to happen.

I have spoken to Olympus about getting the 25x lens but unfortunately there are two problems. 1. it is scarily expensive 2. our system would have to be changed a bit to accommodate it (more expense). I would be curious to see any comparisons if anyone has them, maybe it could be worth the expense:) That being said though I am very, very happy with the quality we are getting from the 20x lens.

Cheers


Cam


Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm


________________________________

From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Fri 29/05/2009 5:25 AM

To: [hidden email]

Subject: Re: Recommendations for commercial multi-photon system purchase

=0 AOn 26/05/2009, at 4:15 AM, Stephen Cody wrote:

> It is true that the upright microscope multiphoton system at the
> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
> lens. This was done at installation by fitting and testing with a
> custom set of lenses for the lens of our choice. I just want to
> clarify that Olympus could always optimise their system for any
> objective at the time of installation. I think from memory I chose
> the 60x WI with cover slip correction for the inverted.


So, the question still stands - has anyone directly tested the older
20x dipping lens (with tuning to fill the back aperture) against the
new 25x? (and is allowed to share the data?)

Apparently a theoretical question for those of with existing systems
but of interest none the less :)

Steve (or Cameron) - you might also be able to comment of the
performance of the Olympus system with the pre-chirp on the DeepSee on/
off (assuming you can turn the compensation off?)


Regards,
Adrian Smith, Centenary Institute

--
Stephen H. Cody

---

Click here to get the very best of AOL, including news, sport, gossip, lifestyles updates and email.

[Craig Brideau]

We have a similar open-prism pair setup.  It is less convenient than
an off-the-shelf automated system but as Evangelos has found it is
easier to deal with changing objectives and optical setups; the
automated systems tend to use look-up tables for specific
lenses/conditions so if you try something new you either need a way to
create a new table entry or have some method for manually tweaking the
nearest table setting.  I had a paper circulating on the list a while
ago about the prism pair setup that some of you may have received.
You can find it as part of the SPIE proceedings for Photonics West
2009.

Craig


On Sat, May 30, 2009 at 6:59 AM, Evangelos
Gatzogiannis<[hidden email]> wrote:

>    There were some good comments on chirp and pre-chirping an IR pulse for
> multiphoton applications.  I don’t have a DeepSee or Chameleon Vision, but
> have/had older Tsunami oscillators and Coherent Mira oscillators.  In both
> cases I’ve built my own pre-chirp prism pair assemblies.  There is no
> choice, in my opinion, between no pre-chirp an chirping.   Some of the
> objective lenses from several manufacturers such as 20x that have less glass
> don’t chirp IR pulses as much if you are operating greater than ~100fs.  You
> must, however, compensate for system optics if you are using 60x or 100x oil
> objectives and going through thick samples.  I have microscopes and
> objectives from Zeiss, Olympus, and Leica and there are differences –
> adjustments to a pre-chirp prism pair must be made daily in my lab to
> compensate for beam pointing stability, to address the different samples my
> users have, and to address the different objective lenses and microscopes my
> users work on.   I like having an open accessible prism pair assembly
> because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300
> confocal and between objectives, scanhead optics, and samples, I find that I
> have to adjust this assembly frequently to optimize the image on a variety
> of samples.  Also, if damage to your sample is a serious problem, and if you
> have significant signal – get a Pockel’s cell or similar modulator.  The
> 76Mhz repetition rate of commercial laser oscillators is like a machine-gun
> operating away, sometimes a hundreds of kHz – 1-2 Mhz rep rate can be
> better.  My regards to all you out there with the Mai Tai Deep See, great
> laser, you can’t do better.  The marriage between  a  Mai Tai Depp See and
> an FV1000 is a very good one.
>
>
>
> Good day,
>
>
>
> Evangelos Gatzogiannis
>
> Ad
>
>
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Stephen Cody
> Sent: Friday, May 29, 2009 10:49 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system purchase
>
>
>
> G'day Adrian,
>
>
>
> As I understand it, the Olympus special 25x dipping objective for
> multiphoton was only available in the US when we ordered and installed the
> Ludwig system (Melbourne, Australia). I tried in vein to get the 25X
> objective. I think it may now be available in Australia, but even so it
> would require a refit of the beam expander to install on the Ludwig system.
> Olympus are very precise when installing, customising and testing of their
> beam expander. It is definitely the most labour intensive part of the
> installation. They not only ensure that the beam fills the back focal plane
> of the chosen objective, they measure the beam intensity profile across the
> beam at this plane, and make adjustments to the lens set in the beam
> expander kit to maximise performance. This data is recorded and sent back to
> Tokyo for approval (well that's what seemed to be happening, I could be
> mistaken). So sadly such a comparison of lenses is not feasible. If and when
> someone has the motorised beam expander this should be a trivial test.
>
>
>
> A similar story with the DeepSee. The pre-chirp optics are always in the
> path of the laser, there is no pre-chirp bypass. I think this is perhaps a
> project that would be more suited to an optics lab, rather than an
> instrument built to investigate biology. Testing the benefit of a pre-chirp
> would take a system such as this offline for at least a couple of months and
> would also require many, many hours of labour by the Olympus engineer to
> reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp
> would require dismantling the the DeepSee itself and reinstalling the IR
> laser, and then repeating the installation process again to get back to the
> original configuration. Unfortunately not a practical thing to test. What I
> can tell you is that it is a very simple matter to adjust the the pre-chirp
> with your specimen to obtain the brightest signal. A poorly set pre-chirp
> compared to an adjusted pre-chirp makes quite a difference. But without
> knowing what the scenario would be with no pre-chirp it is very difficult to
> give a precise answer.
>
>
>
> My gut feeling is pre-chirping is fantastic, although Cameron has certainly
> used it much more than I, so I deffer to him in this regard. Logically, if
> you can see a clear benefit of adjusting the pre-chirp (more fluorescence
> and so laser power can be reduced). One would think that this must be better
> than a system with no pre-chirp where the pulse is spread ("chirped") by the
> optics of the microscope, and cannot be compensated. I don't want to get
> into brand name comparisons but I think it important to to let people know
> that (as explained to me) It seems that different microscope manufacturers
> have markedly different amounts of chirp associated with their Optics. So
> the decision "to Pre-Chirp or not to Pre-Chirp" is far more important
> depending on the brand of microscope you decide to go with. As this was the
> first installation of its kind, little was known by anyone at the time what
> was required. It turned out that originally the DeepSee imparted too much
> correction for our Olympus scope, and SpectraPhysics very quickly supplied a
> "Low Dispersion" modification to the setup. After the modification we could
> find the "sweet spot" with the DeepSee pre-chirp extremely easily.
>
>
>
> The point being with at least some other brands, pre-chirping is probably
> much more important. I would ignore the "company speak" that maintains that
> a similar benefit can be achieved simply by increasing IR laser power. I'm
> not sure if the Multiphoton List is still going (I think it's membership was
> merged with this list). However the subject "to Pre-Chirp or not to
> Pre-Chirp" was raised by myself on that list a couple of years ago. Someone
> replied to me (I think personally not via the list), that they demonstrated
> a system: Without pre-chirp they observed damage to their tissue in a whole
> mouse, above and below the plane of focus, but the fluorophore at the plane
> of focus was not bleached. This suggested direct IR damage of the laser, but
> that the 2P effect at the fluorophore was not damaging. When the pre-chirp
> was installed (or perhaps it was adjusted correctly) this direct IR damage
> was not seen, presumably lower laser power could be used, to achieve the
> multiphoton effect. I'm sorry I can't remember who gave me this information
> and I don't have access to my old email anymore. But a big thank you, that
> bit of information was exactly what I needed to make a decision and allowed
> me to cut through the "company speak".
>
>
>
> I would imagine that a collaboration with SpectraPhysics and a microscope
> company, so that the pre-chirp could be bypassed would be the best way
> forward to test this. Does SpectraPhysics and a microscope company want to
> loan me a multiphoton for 12 months (or 12 years for that matter)? I have
> not mentioned Coherent, at the time they were only offering a third party
> pre-chirp option. However, Coherent now offer the "Chameleon Vision" with
> integrated pre-chirp, this too should be considered in any MPE purchase
> decision.
>
>
>
> I have no commercial affiliation with any of the companies mentioned.
>
>
>
> Cheers
>
> Steve
>
>
>
> Stephen H. Cody
> Imaging Research Fellow & Manager
> Monash Micro Imaging – AMREP
> Monash University
> 6 Floor Burnet Tower
> Alfred Medical Research & Education Precinct
> 89 Commercial Rd, Melbourne, Australia, 3004
>
> www.microimaging.monash.org
> Phone (Monash):  (613) 990 30142
> Phone (BakerIDI): (613) 8532 1580
>
>
>
> 2009/5/29 Cameron Nowell <[hidden email]>
>
> Hi Adrian,
>
> After just having a brief look I don't think the pre-chirp unit can be
> disabled, there is no off switch and I am pretty sure if I disconnect the
> control box all sorts of weird things will go wrong. There is already one
> redundant component on the Maintain system that can not be removed for that
> reason.
>
> I can say that if it is not set optimally there is a reduction in image
> quality and penetration depth. Although it does seem very dependent on the
> sample and the fluorophore. On some samples you can gain an extra 100 or
> more micron in the depth by tuning the pre-chirp. Other samples not a lot
> seems to happen.
>
> I have spoken to Olympus about getting the 25x lens but unfortunately there
> are two problems. 1. it is scarily expensive 2. our system would have to be
> changed a bit to accommodate it (more expense). I would be curious to see
> any comparisons if anyone has them, maybe it could be worth the expense:)
> That being said though I am very, very happy with the quality we are getting
> from the 20x lens.
>
> Cheers
>
>
> Cam
>
>
> Cameron J. Nowell
> Microscpy Manager
> Central Resource for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
>
> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Adrian Smith
> Sent: Fri 29/05/2009 5:25 AM
>
> To: [hidden email]
>
> Subject: Re: Recommendations for commercial multi-photon system purchase
>
>
> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:
>
>> It is true that the upright microscope multiphoton system at the
>> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
>> lens. This was done at installation by fitting and testing with a
>> custom set of lenses for the lens of our choice. I just want to
>> clarify that Olympus could always optimise their system for any
>> objective at the time of installation. I think from memory I chose
>> the 60x WI with cover slip correction for the inverted.
>
>
> So, the question still stands - has anyone directly tested the older
> 20x dipping lens (with tuning to fill the back aperture) against the
> new 25x? (and is allowed to share the data?)
>
> Apparently a theoretical question for those of with existing systems
> but of interest none the less :)
>
> Steve (or Cameron) - you might also be able to comment of the
> performance of the Olympus system with the pre-chirp on the DeepSee on/
> off (assuming you can turn the compensation off?)
>
>
> Regards,
> Adrian Smith, Centenary Institute
>
>
> --
> Stephen H. Cody

[Armstrong, Brian]

I do remember looking at your set-up. Approximately how far do you have to move your prisms (in extreme cases, say you are going from a 5x to a 20x/0.95)?
I always thought that you had to change the inter-prism distance an appreciable amount and wonder how they (Spectra and Coherent) accomplish this is such a small box.
Thanks in advance,

Brian Armstrong PhD
Manager, Light Microscopy Core
Beckman Research Institute
1450 East Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
 
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, June 02, 2009 10:14 AM
To: [hidden email]
Subject: Re: Chirp

We have a similar open-prism pair setup.  It is less convenient than
an off-the-shelf automated system but as Evangelos has found it is
easier to deal with changing objectives and optical setups; the
automated systems tend to use look-up tables for specific
lenses/conditions so if you try something new you either need a way to
create a new table entry or have some method for manually tweaking the
nearest table setting.  I had a paper circulating on the list a while
ago about the prism pair setup that some of you may have received.
You can find it as part of the SPIE proceedings for Photonics West
2009.

Craig


On Sat, May 30, 2009 at 6:59 AM, Evangelos
Gatzogiannis<[hidden email]> wrote:

>    There were some good comments on chirp and pre-chirping an IR pulse for
> multiphoton applications.  I don't have a DeepSee or Chameleon Vision, but
> have/had older Tsunami oscillators and Coherent Mira oscillators.  In both
> cases I've built my own pre-chirp prism pair assemblies.  There is no
> choice, in my opinion, between no pre-chirp an chirping.   Some of the
> objective lenses from several manufacturers such as 20x that have less glass
> don't chirp IR pulses as much if you are operating greater than ~100fs.  You
> must, however, compensate for system optics if you are using 60x or 100x oil
> objectives and going through thick samples.  I have microscopes and
> objectives from Zeiss, Olympus, and Leica and there are differences -
> adjustments to a pre-chirp prism pair must be made daily in my lab to
> compensate for beam pointing stability, to address the different samples my
> users have, and to address the different objective lenses and microscopes my
> users work on.   I like having an open accessible prism pair assembly
> because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300
> confocal and between objectives, scanhead optics, and samples, I find that I
> have to adjust this assembly frequently to optimize the image on a variety
> of samples.  Also, if damage to your sample is a serious problem, and if you
> have significant signal - get a Pockel's cell or similar modulator.  The
> 76Mhz repetition rate of commercial laser oscillators is like a machine-gun
> operating away, sometimes a hundreds of kHz - 1-2 Mhz rep rate can be
> better.  My regards to all you out there with the Mai Tai Deep See, great
> laser, you can't do better.  The marriage between  a  Mai Tai Depp See and
> an FV1000 is a very good one.
>
>
>
> Good day,
>
>
>
> Evangelos Gatzogiannis
>
> Ad
>
>
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Stephen Cody
> Sent: Friday, May 29, 2009 10:49 PM
> To: [hidden email]
> Subject: Re: Recommendations for commercial multi-photon system purchase
>
>
>
> G'day Adrian,
>
>
>
> As I understand it, the Olympus special 25x dipping objective for
> multiphoton was only available in the US when we ordered and installed the
> Ludwig system (Melbourne, Australia). I tried in vein to get the 25X
> objective. I think it may now be available in Australia, but even so it
> would require a refit of the beam expander to install on the Ludwig system.
> Olympus are very precise when installing, customising and testing of their
> beam expander. It is definitely the most labour intensive part of the
> installation. They not only ensure that the beam fills the back focal plane
> of the chosen objective, they measure the beam intensity profile across the
> beam at this plane, and make adjustments to the lens set in the beam
> expander kit to maximise performance. This data is recorded and sent back to
> Tokyo for approval (well that's what seemed to be happening, I could be
> mistaken). So sadly such a comparison of lenses is not feasible. If and when
> someone has the motorised beam expander this should be a trivial test.
>
>
>
> A similar story with the DeepSee. The pre-chirp optics are always in the
> path of the laser, there is no pre-chirp bypass. I think this is perhaps a
> project that would be more suited to an optics lab, rather than an
> instrument built to investigate biology. Testing the benefit of a pre-chirp
> would take a system such as this offline for at least a couple of months and
> would also require many, many hours of labour by the Olympus engineer to
> reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp
> would require dismantling the the DeepSee itself and reinstalling the IR
> laser, and then repeating the installation process again to get back to the
> original configuration. Unfortunately not a practical thing to test. What I
> can tell you is that it is a very simple matter to adjust the the pre-chirp
> with your specimen to obtain the brightest signal. A poorly set pre-chirp
> compared to an adjusted pre-chirp makes quite a difference. But without
> knowing what the scenario would be with no pre-chirp it is very difficult to
> give a precise answer.
>
>
>
> My gut feeling is pre-chirping is fantastic, although Cameron has certainly
> used it much more than I, so I deffer to him in this regard. Logically, if
> you can see a clear benefit of adjusting the pre-chirp (more fluorescence
> and so laser power can be reduced). One would think that this must be better
> than a system with no pre-chirp where the pulse is spread ("chirped") by the
> optics of the microscope, and cannot be compensated. I don't want to get
> into brand name comparisons but I think it important to to let people know
> that (as explained to me) It seems that different microscope manufacturers
> have markedly different amounts of chirp associated with their Optics. So
> the decision "to Pre-Chirp or not to Pre-Chirp" is far more important
> depending on the brand of microscope you decide to go with. As this was the
> first installation of its kind, little was known by anyone at the time what
> was required. It turned out that originally the DeepSee imparted too much
> correction for our Olympus scope, and SpectraPhysics very quickly supplied a
> "Low Dispersion" modification to the setup. After the modification we could
> find the "sweet spot" with the DeepSee pre-chirp extremely easily.
>
>
>
> The point being with at least some other brands, pre-chirping is probably
> much more important. I would ignore the "company speak" that maintains that
> a similar benefit can be achieved simply by increasing IR laser power. I'm
> not sure if the Multiphoton List is still going (I think it's membership was
> merged with this list). However the subject "to Pre-Chirp or not to
> Pre-Chirp" was raised by myself on that list a couple of years ago. Someone
> replied to me (I think personally not via the list), that they demonstrated
> a system: Without pre-chirp they observed damage to their tissue in a whole
> mouse, above and below the plane of focus, but the fluorophore at the plane
> of focus was not bleached. This suggested direct IR damage of the laser, but
> that the 2P effect at the fluorophore was not damaging. When the pre-chirp
> was installed (or perhaps it was adjusted correctly) this direct IR damage
> was not seen, presumably lower laser power could be used, to achieve the
> multiphoton effect. I'm sorry I can't remember who gave me this information
> and I don't have access to my old email anymore. But a big thank you, that
> bit of information was exactly what I needed to make a decision and allowed
> me to cut through the "company speak".
>
>
>
> I would imagine that a collaboration with SpectraPhysics and a microscope
> company, so that the pre-chirp could be bypassed would be the best way
> forward to test this. Does SpectraPhysics and a microscope company want to
> loan me a multiphoton for 12 months (or 12 years for that matter)? I have
> not mentioned Coherent, at the time they were only offering a third party
> pre-chirp option. However, Coherent now offer the "Chameleon Vision" with
> integrated pre-chirp, this too should be considered in any MPE purchase
> decision.
>
>
>
> I have no commercial affiliation with any of the companies mentioned.
>
>
>
> Cheers
>
> Steve
>
>
>
> Stephen H. Cody
> Imaging Research Fellow & Manager
> Monash Micro Imaging - AMREP
> Monash University
> 6 Floor Burnet Tower
> Alfred Medical Research & Education Precinct
> 89 Commercial Rd, Melbourne, Australia, 3004
>
> www.microimaging.monash.org
> Phone (Monash):  (613) 990 30142
> Phone (BakerIDI): (613) 8532 1580
>
>
>
> 2009/5/29 Cameron Nowell <[hidden email]>
>
> Hi Adrian,
>
> After just having a brief look I don't think the pre-chirp unit can be
> disabled, there is no off switch and I am pretty sure if I disconnect the
> control box all sorts of weird things will go wrong. There is already one
> redundant component on the Maintain system that can not be removed for that
> reason.
>
> I can say that if it is not set optimally there is a reduction in image
> quality and penetration depth. Although it does seem very dependent on the
> sample and the fluorophore. On some samples you can gain an extra 100 or
> more micron in the depth by tuning the pre-chirp. Other samples not a lot
> seems to happen.
>
> I have spoken to Olympus about getting the 25x lens but unfortunately there
> are two problems. 1. it is scarily expensive 2. our system would have to be
> changed a bit to accommodate it (more expense). I would be curious to see
> any comparisons if anyone has them, maybe it could be worth the expense:)
> That being said though I am very, very happy with the quality we are getting
> from the 20x lens.
>
> Cheers
>
>
> Cam
>
>
> Cameron J. Nowell
> Microscpy Manager
> Central Resource for Advanced Microscopy
> Ludwig Insttue for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
>
> Office: [phone]
> Mobile: [phone]
> Fax: [phone]
>
> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>
>
> ________________________________
>
> From: Confocal Microscopy List on behalf of Adrian Smith
> Sent: Fri 29/05/2009 5:25 AM
>
> To: [hidden email]
>
> Subject: Re: Recommendations for commercial multi-photon system purchase
>
>
> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:
>
>> It is true that the upright microscope multiphoton system at the
>> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
>> lens. This was done at installation by fitting and testing with a
>> custom set of lenses for the lens of our choice. I just want to
>> clarify that Olympus could always optimise their system for any
>> objective at the time of installation. I think from memory I chose
>> the 60x WI with cover slip correction for the inverted.
>
>
> So, the question still stands - has anyone directly tested the older
> 20x dipping lens (with tuning to fill the back aperture) against the
> new 25x? (and is allowed to share the data?)
>
> Apparently a theoretical question for those of with existing systems
> but of interest none the less :)
>
> Steve (or Cameron) - you might also be able to comment of the
> performance of the Olympus system with the pre-chirp on the DeepSee on/
> off (assuming you can turn the compensation off?)
>
>
> Regards,
> Adrian Smith, Centenary Institute
>
>
> --
> Stephen H. Cody

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[Craig Brideau]

There are two tricks; use a very high index glass in the prisms, and
fold your design with mirrors.  My design does both of these things,
although the commercial designs use even more mirror folding to
shorten the physical distance between the prisms.  Some designs only
use a single larger prism and just fold back into it repeatedly.  The
downside to folding is you have to be careful with your alignment,
although using retroreflectors rather than flat mirrors minimizes this
as they are less sensitive to misalignment.  Using high index glass
also helps considerably, although if your pulse bandwidth is too great
(greater than about 15nm) then you can start having higher-order
dispersion issues.  Our laser is an older Spectra Physics Tsunami
model; you can vary the spectral bandwidth of the pulse by manually
adjusting the laser, so we can get optimal bandwidth and dispersion
combinations with some manual tweaking of the pulse compressor and the
laser together.  It takes about 15-30 minutes to do a wavelength
re-tune with the system we've set up (shorter if we are tuning visible
wavelengths as those are easier to work with).  Dispersion adjustment
once the wavelength is set takes about 1 minute.  I can compensate for
our standard Nikon C1 microscope and 60x dipping lens with about
10-20cm of path length even with ~15nm spectral bandwidth in the
pulses with only a little residual 3rd order distortion.  The temporal
pulse width at the sample is probably around 60-70fs with best
adjustment.  I have also done some experiments with creating strong
chirps in pulses on purpose.  With about 1m of separation I can chirp
a 15nm spectrally wide pulse out to about 2ps (negative chirp).  My
compressor design can also be set to a 'neutral' position (path length
~5cm) where it has no effect (still some very small residual 3rd order
if bandwidth is high) on the pulses.  If I bring the prisms to their
minimum distance (a couple cm) I can actually get 100-150fs positively
chirped pulses out of the compressor from 60fs pulses.  Here the high
index of the glass causes enough positive chirping that it actually
overwhelms the geometric path-length adjustment that produces negative
chirp.  Fun times!  @:-)

Craig


On Tue, Jun 2, 2009 at 11:54 AM, Armstrong, Brian<[hidden email]> wrote:

> I do remember looking at your set-up. Approximately how far do you have to move your prisms (in extreme cases, say you are going from a 5x to a 20x/0.95)?
> I always thought that you had to change the inter-prism distance an appreciable amount and wonder how they (Spectra and Coherent) accomplish this is such a small box.
> Thanks in advance,
>
> Brian Armstrong PhD
> Manager, Light Microscopy Core
> Beckman Research Institute
> 1450 East Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
> http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Tuesday, June 02, 2009 10:14 AM
> To: [hidden email]
> Subject: Re: Chirp
>
> We have a similar open-prism pair setup.  It is less convenient than
> an off-the-shelf automated system but as Evangelos has found it is
> easier to deal with changing objectives and optical setups; the
> automated systems tend to use look-up tables for specific
> lenses/conditions so if you try something new you either need a way to
> create a new table entry or have some method for manually tweaking the
> nearest table setting.  I had a paper circulating on the list a while
> ago about the prism pair setup that some of you may have received.
> You can find it as part of the SPIE proceedings for Photonics West
> 2009.
>
> Craig
>
>
> On Sat, May 30, 2009 at 6:59 AM, Evangelos
> Gatzogiannis<[hidden email]> wrote:
>>    There were some good comments on chirp and pre-chirping an IR pulse for
>> multiphoton applications.  I don't have a DeepSee or Chameleon Vision, but
>> have/had older Tsunami oscillators and Coherent Mira oscillators.  In both
>> cases I've built my own pre-chirp prism pair assemblies.  There is no
>> choice, in my opinion, between no pre-chirp an chirping.   Some of the
>> objective lenses from several manufacturers such as 20x that have less glass
>> don't chirp IR pulses as much if you are operating greater than ~100fs.  You
>> must, however, compensate for system optics if you are using 60x or 100x oil
>> objectives and going through thick samples.  I have microscopes and
>> objectives from Zeiss, Olympus, and Leica and there are differences -
>> adjustments to a pre-chirp prism pair must be made daily in my lab to
>> compensate for beam pointing stability, to address the different samples my
>> users have, and to address the different objective lenses and microscopes my
>> users work on.   I like having an open accessible prism pair assembly
>> because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300
>> confocal and between objectives, scanhead optics, and samples, I find that I
>> have to adjust this assembly frequently to optimize the image on a variety
>> of samples.  Also, if damage to your sample is a serious problem, and if you
>> have significant signal - get a Pockel's cell or similar modulator.  The
>> 76Mhz repetition rate of commercial laser oscillators is like a machine-gun
>> operating away, sometimes a hundreds of kHz - 1-2 Mhz rep rate can be
>> better.  My regards to all you out there with the Mai Tai Deep See, great
>> laser, you can't do better.  The marriage between  a  Mai Tai Depp See and
>> an FV1000 is a very good one.
>>
>>
>>
>> Good day,
>>
>>
>>
>> Evangelos Gatzogiannis
>>
>> Ad
>>
>>
>>
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Stephen Cody
>> Sent: Friday, May 29, 2009 10:49 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>>
>> G'day Adrian,
>>
>>
>>
>> As I understand it, the Olympus special 25x dipping objective for
>> multiphoton was only available in the US when we ordered and installed the
>> Ludwig system (Melbourne, Australia). I tried in vein to get the 25X
>> objective. I think it may now be available in Australia, but even so it
>> would require a refit of the beam expander to install on the Ludwig system.
>> Olympus are very precise when installing, customising and testing of their
>> beam expander. It is definitely the most labour intensive part of the
>> installation. They not only ensure that the beam fills the back focal plane
>> of the chosen objective, they measure the beam intensity profile across the
>> beam at this plane, and make adjustments to the lens set in the beam
>> expander kit to maximise performance. This data is recorded and sent back to
>> Tokyo for approval (well that's what seemed to be happening, I could be
>> mistaken). So sadly such a comparison of lenses is not feasible. If and when
>> someone has the motorised beam expander this should be a trivial test.
>>
>>
>>
>> A similar story with the DeepSee. The pre-chirp optics are always in the
>> path of the laser, there is no pre-chirp bypass. I think this is perhaps a
>> project that would be more suited to an optics lab, rather than an
>> instrument built to investigate biology. Testing the benefit of a pre-chirp
>> would take a system such as this offline for at least a couple of months and
>> would also require many, many hours of labour by the Olympus engineer to
>> reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp
>> would require dismantling the the DeepSee itself and reinstalling the IR
>> laser, and then repeating the installation process again to get back to the
>> original configuration. Unfortunately not a practical thing to test. What I
>> can tell you is that it is a very simple matter to adjust the the pre-chirp
>> with your specimen to obtain the brightest signal. A poorly set pre-chirp
>> compared to an adjusted pre-chirp makes quite a difference. But without
>> knowing what the scenario would be with no pre-chirp it is very difficult to
>> give a precise answer.
>>
>>
>>
>> My gut feeling is pre-chirping is fantastic, although Cameron has certainly
>> used it much more than I, so I deffer to him in this regard. Logically, if
>> you can see a clear benefit of adjusting the pre-chirp (more fluorescence
>> and so laser power can be reduced). One would think that this must be better
>> than a system with no pre-chirp where the pulse is spread ("chirped") by the
>> optics of the microscope, and cannot be compensated. I don't want to get
>> into brand name comparisons but I think it important to to let people know
>> that (as explained to me) It seems that different microscope manufacturers
>> have markedly different amounts of chirp associated with their Optics. So
>> the decision "to Pre-Chirp or not to Pre-Chirp" is far more important
>> depending on the brand of microscope you decide to go with. As this was the
>> first installation of its kind, little was known by anyone at the time what
>> was required. It turned out that originally the DeepSee imparted too much
>> correction for our Olympus scope, and SpectraPhysics very quickly supplied a
>> "Low Dispersion" modification to the setup. After the modification we could
>> find the "sweet spot" with the DeepSee pre-chirp extremely easily.
>>
>>
>>
>> The point being with at least some other brands, pre-chirping is probably
>> much more important. I would ignore the "company speak" that maintains that
>> a similar benefit can be achieved simply by increasing IR laser power. I'm
>> not sure if the Multiphoton List is still going (I think it's membership was
>> merged with this list). However the subject "to Pre-Chirp or not to
>> Pre-Chirp" was raised by myself on that list a couple of years ago. Someone
>> replied to me (I think personally not via the list), that they demonstrated
>> a system: Without pre-chirp they observed damage to their tissue in a whole
>> mouse, above and below the plane of focus, but the fluorophore at the plane
>> of focus was not bleached. This suggested direct IR damage of the laser, but
>> that the 2P effect at the fluorophore was not damaging. When the pre-chirp
>> was installed (or perhaps it was adjusted correctly) this direct IR damage
>> was not seen, presumably lower laser power could be used, to achieve the
>> multiphoton effect. I'm sorry I can't remember who gave me this information
>> and I don't have access to my old email anymore. But a big thank you, that
>> bit of information was exactly what I needed to make a decision and allowed
>> me to cut through the "company speak".
>>
>>
>>
>> I would imagine that a collaboration with SpectraPhysics and a microscope
>> company, so that the pre-chirp could be bypassed would be the best way
>> forward to test this. Does SpectraPhysics and a microscope company want to
>> loan me a multiphoton for 12 months (or 12 years for that matter)? I have
>> not mentioned Coherent, at the time they were only offering a third party
>> pre-chirp option. However, Coherent now offer the "Chameleon Vision" with
>> integrated pre-chirp, this too should be considered in any MPE purchase
>> decision.
>>
>>
>>
>> I have no commercial affiliation with any of the companies mentioned.
>>
>>
>>
>> Cheers
>>
>> Steve
>>
>>
>>
>> Stephen H. Cody
>> Imaging Research Fellow & Manager
>> Monash Micro Imaging - AMREP
>> Monash University
>> 6 Floor Burnet Tower
>> Alfred Medical Research & Education Precinct
>> 89 Commercial Rd, Melbourne, Australia, 3004
>>
>> www.microimaging.monash.org
>> Phone (Monash):  (613) 990 30142
>> Phone (BakerIDI): (613) 8532 1580
>>
>>
>>
>> 2009/5/29 Cameron Nowell <[hidden email]>
>>
>> Hi Adrian,
>>
>> After just having a brief look I don't think the pre-chirp unit can be
>> disabled, there is no off switch and I am pretty sure if I disconnect the
>> control box all sorts of weird things will go wrong. There is already one
>> redundant component on the Maintain system that can not be removed for that
>> reason.
>>
>> I can say that if it is not set optimally there is a reduction in image
>> quality and penetration depth. Although it does seem very dependent on the
>> sample and the fluorophore. On some samples you can gain an extra 100 or
>> more micron in the depth by tuning the pre-chirp. Other samples not a lot
>> seems to happen.
>>
>> I have spoken to Olympus about getting the 25x lens but unfortunately there
>> are two problems. 1. it is scarily expensive 2. our system would have to be
>> changed a bit to accommodate it (more expense). I would be curious to see
>> any comparisons if anyone has them, maybe it could be worth the expense:)
>> That being said though I am very, very happy with the quality we are getting
>> from the 20x lens.
>>
>> Cheers
>>
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscpy Manager
>> Central Resource for Advanced Microscopy
>> Ludwig Insttue for Cancer Research
>> PO Box 2008
>> Royal Melbourne Hospital
>> Victoria, 3050
>> AUSTRALIA
>>
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>>
>> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>>
>>
>> ________________________________
>>
>> From: Confocal Microscopy List on behalf of Adrian Smith
>> Sent: Fri 29/05/2009 5:25 AM
>>
>> To: [hidden email]
>>
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:
>>
>>> It is true that the upright microscope multiphoton system at the
>>> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
>>> lens. This was done at installation by fitting and testing with a
>>> custom set of lenses for the lens of our choice. I just want to
>>> clarify that Olympus could always optimise their system for any
>>> objective at the time of installation. I think from memory I chose
>>> the 60x WI with cover slip correction for the inverted.
>>
>>
>> So, the question still stands - has anyone directly tested the older
>> 20x dipping lens (with tuning to fill the back aperture) against the
>> new 25x? (and is allowed to share the data?)
>>
>> Apparently a theoretical question for those of with existing systems
>> but of interest none the less :)
>>
>> Steve (or Cameron) - you might also be able to comment of the
>> performance of the Olympus system with the pre-chirp on the DeepSee on/
>> off (assuming you can turn the compensation off?)
>>
>>
>> Regards,
>> Adrian Smith, Centenary Institute
>>
>>
>> --
>> Stephen H. Cody
>
>
> ---------------------------------------------------------------------
>
> SECURITY/CONFIDENTIALITY WARNING:
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>

[Armstrong, Brian]

Thanks for the info. I figured it would be accomplished through folding. I did not think about the high index glass.
Still, I think 20cm seems like a long way to move these prisms, especially if you are doing it a lot. Is the tuning consistently repeatable with the Deep See and Coherent systems? Anyone care to comment?
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, June 02, 2009 11:40 AM
To: [hidden email]
Subject: Re: Chirp

There are two tricks; use a very high index glass in the prisms, and
fold your design with mirrors.  My design does both of these things,
although the commercial designs use even more mirror folding to
shorten the physical distance between the prisms.  Some designs only
use a single larger prism and just fold back into it repeatedly.  The
downside to folding is you have to be careful with your alignment,
although using retroreflectors rather than flat mirrors minimizes this
as they are less sensitive to misalignment.  Using high index glass
also helps considerably, although if your pulse bandwidth is too great
(greater than about 15nm) then you can start having higher-order
dispersion issues.  Our laser is an older Spectra Physics Tsunami
model; you can vary the spectral bandwidth of the pulse by manually
adjusting the laser, so we can get optimal bandwidth and dispersion
combinations with some manual tweaking of the pulse compressor and the
laser together.  It takes about 15-30 minutes to do a wavelength
re-tune with the system we've set up (shorter if we are tuning visible
wavelengths as those are easier to work with).  Dispersion adjustment
once the wavelength is set takes about 1 minute.  I can compensate for
our standard Nikon C1 microscope and 60x dipping lens with about
10-20cm of path length even with ~15nm spectral bandwidth in the
pulses with only a little residual 3rd order distortion.  The temporal
pulse width at the sample is probably around 60-70fs with best
adjustment.  I have also done some experiments with creating strong
chirps in pulses on purpose.  With about 1m of separation I can chirp
a 15nm spectrally wide pulse out to about 2ps (negative chirp).  My
compressor design can also be set to a 'neutral' position (path length
~5cm) where it has no effect (still some very small residual 3rd order
if bandwidth is high) on the pulses.  If I bring the prisms to their
minimum distance (a couple cm) I can actually get 100-150fs positively
chirped pulses out of the compressor from 60fs pulses.  Here the high
index of the glass causes enough positive chirping that it actually
overwhelms the geometric path-length adjustment that produces negative
chirp.  Fun times!  @:-)

Craig


On Tue, Jun 2, 2009 at 11:54 AM, Armstrong, Brian<[hidden email]> wrote:

> I do remember looking at your set-up. Approximately how far do you have to move your prisms (in extreme cases, say you are going from a 5x to a 20x/0.95)?
> I always thought that you had to change the inter-prism distance an appreciable amount and wonder how they (Spectra and Coherent) accomplish this is such a small box.
> Thanks in advance,
>
> Brian Armstrong PhD
> Manager, Light Microscopy Core
> Beckman Research Institute
> 1450 East Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
> http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Tuesday, June 02, 2009 10:14 AM
> To: [hidden email]
> Subject: Re: Chirp
>
> We have a similar open-prism pair setup.  It is less convenient than
> an off-the-shelf automated system but as Evangelos has found it is
> easier to deal with changing objectives and optical setups; the
> automated systems tend to use look-up tables for specific
> lenses/conditions so if you try something new you either need a way to
> create a new table entry or have some method for manually tweaking the
> nearest table setting.  I had a paper circulating on the list a while
> ago about the prism pair setup that some of you may have received.
> You can find it as part of the SPIE proceedings for Photonics West
> 2009.
>
> Craig
>
>
> On Sat, May 30, 2009 at 6:59 AM, Evangelos
> Gatzogiannis<[hidden email]> wrote:
>>    There were some good comments on chirp and pre-chirping an IR pulse for
>> multiphoton applications.  I don't have a DeepSee or Chameleon Vision, but
>> have/had older Tsunami oscillators and Coherent Mira oscillators.  In both
>> cases I've built my own pre-chirp prism pair assemblies.  There is no
>> choice, in my opinion, between no pre-chirp an chirping.   Some of the
>> objective lenses from several manufacturers such as 20x that have less glass
>> don't chirp IR pulses as much if you are operating greater than ~100fs.  You
>> must, however, compensate for system optics if you are using 60x or 100x oil
>> objectives and going through thick samples.  I have microscopes and
>> objectives from Zeiss, Olympus, and Leica and there are differences -
>> adjustments to a pre-chirp prism pair must be made daily in my lab to
>> compensate for beam pointing stability, to address the different samples my
>> users have, and to address the different objective lenses and microscopes my
>> users work on.   I like having an open accessible prism pair assembly
>> because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300
>> confocal and between objectives, scanhead optics, and samples, I find that I
>> have to adjust this assembly frequently to optimize the image on a variety
>> of samples.  Also, if damage to your sample is a serious problem, and if you
>> have significant signal - get a Pockel's cell or similar modulator.  The
>> 76Mhz repetition rate of commercial laser oscillators is like a machine-gun
>> operating away, sometimes a hundreds of kHz - 1-2 Mhz rep rate can be
>> better.  My regards to all you out there with the Mai Tai Deep See, great
>> laser, you can't do better.  The marriage between  a  Mai Tai Depp See and
>> an FV1000 is a very good one.
>>
>>
>>
>> Good day,
>>
>>
>>
>> Evangelos Gatzogiannis
>>
>> Ad
>>
>>
>>
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Stephen Cody
>> Sent: Friday, May 29, 2009 10:49 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>>
>> G'day Adrian,
>>
>>
>>
>> As I understand it, the Olympus special 25x dipping objective for
>> multiphoton was only available in the US when we ordered and installed the
>> Ludwig system (Melbourne, Australia). I tried in vein to get the 25X
>> objective. I think it may now be available in Australia, but even so it
>> would require a refit of the beam expander to install on the Ludwig system.
>> Olympus are very precise when installing, customising and testing of their
>> beam expander. It is definitely the most labour intensive part of the
>> installation. They not only ensure that the beam fills the back focal plane
>> of the chosen objective, they measure the beam intensity profile across the
>> beam at this plane, and make adjustments to the lens set in the beam
>> expander kit to maximise performance. This data is recorded and sent back to
>> Tokyo for approval (well that's what seemed to be happening, I could be
>> mistaken). So sadly such a comparison of lenses is not feasible. If and when
>> someone has the motorised beam expander this should be a trivial test.
>>
>>
>>
>> A similar story with the DeepSee. The pre-chirp optics are always in the
>> path of the laser, there is no pre-chirp bypass. I think this is perhaps a
>> project that would be more suited to an optics lab, rather than an
>> instrument built to investigate biology. Testing the benefit of a pre-chirp
>> would take a system such as this offline for at least a couple of months and
>> would also require many, many hours of labour by the Olympus engineer to
>> reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp
>> would require dismantling the the DeepSee itself and reinstalling the IR
>> laser, and then repeating the installation process again to get back to the
>> original configuration. Unfortunately not a practical thing to test. What I
>> can tell you is that it is a very simple matter to adjust the the pre-chirp
>> with your specimen to obtain the brightest signal. A poorly set pre-chirp
>> compared to an adjusted pre-chirp makes quite a difference. But without
>> knowing what the scenario would be with no pre-chirp it is very difficult to
>> give a precise answer.
>>
>>
>>
>> My gut feeling is pre-chirping is fantastic, although Cameron has certainly
>> used it much more than I, so I deffer to him in this regard. Logically, if
>> you can see a clear benefit of adjusting the pre-chirp (more fluorescence
>> and so laser power can be reduced). One would think that this must be better
>> than a system with no pre-chirp where the pulse is spread ("chirped") by the
>> optics of the microscope, and cannot be compensated. I don't want to get
>> into brand name comparisons but I think it important to to let people know
>> that (as explained to me) It seems that different microscope manufacturers
>> have markedly different amounts of chirp associated with their Optics. So
>> the decision "to Pre-Chirp or not to Pre-Chirp" is far more important
>> depending on the brand of microscope you decide to go with. As this was the
>> first installation of its kind, little was known by anyone at the time what
>> was required. It turned out that originally the DeepSee imparted too much
>> correction for our Olympus scope, and SpectraPhysics very quickly supplied a
>> "Low Dispersion" modification to the setup. After the modification we could
>> find the "sweet spot" with the DeepSee pre-chirp extremely easily.
>>
>>
>>
>> The point being with at least some other brands, pre-chirping is probably
>> much more important. I would ignore the "company speak" that maintains that
>> a similar benefit can be achieved simply by increasing IR laser power. I'm
>> not sure if the Multiphoton List is still going (I think it's membership was
>> merged with this list). However the subject "to Pre-Chirp or not to
>> Pre-Chirp" was raised by myself on that list a couple of years ago. Someone
>> replied to me (I think personally not via the list), that they demonstrated
>> a system: Without pre-chirp they observed damage to their tissue in a whole
>> mouse, above and below the plane of focus, but the fluorophore at the plane
>> of focus was not bleached. This suggested direct IR damage of the laser, but
>> that the 2P effect at the fluorophore was not damaging. When the pre-chirp
>> was installed (or perhaps it was adjusted correctly) this direct IR damage
>> was not seen, presumably lower laser power could be used, to achieve the
>> multiphoton effect. I'm sorry I can't remember who gave me this information
>> and I don't have access to my old email anymore. But a big thank you, that
>> bit of information was exactly what I needed to make a decision and allowed
>> me to cut through the "company speak".
>>
>>
>>
>> I would imagine that a collaboration with SpectraPhysics and a microscope
>> company, so that the pre-chirp could be bypassed would be the best way
>> forward to test this. Does SpectraPhysics and a microscope company want to
>> loan me a multiphoton for 12 months (or 12 years for that matter)? I have
>> not mentioned Coherent, at the time they were only offering a third party
>> pre-chirp option. However, Coherent now offer the "Chameleon Vision" with
>> integrated pre-chirp, this too should be considered in any MPE purchase
>> decision.
>>
>>
>>
>> I have no commercial affiliation with any of the companies mentioned.
>>
>>
>>
>> Cheers
>>
>> Steve
>>
>>
>>
>> Stephen H. Cody
>> Imaging Research Fellow & Manager
>> Monash Micro Imaging - AMREP
>> Monash University
>> 6 Floor Burnet Tower
>> Alfred Medical Research & Education Precinct
>> 89 Commercial Rd, Melbourne, Australia, 3004
>>
>> www.microimaging.monash.org
>> Phone (Monash):  (613) 990 30142
>> Phone (BakerIDI): (613) 8532 1580
>>
>>
>>
>> 2009/5/29 Cameron Nowell <[hidden email]>
>>
>> Hi Adrian,
>>
>> After just having a brief look I don't think the pre-chirp unit can be
>> disabled, there is no off switch and I am pretty sure if I disconnect the
>> control box all sorts of weird things will go wrong. There is already one
>> redundant component on the Maintain system that can not be removed for that
>> reason.
>>
>> I can say that if it is not set optimally there is a reduction in image
>> quality and penetration depth. Although it does seem very dependent on the
>> sample and the fluorophore. On some samples you can gain an extra 100 or
>> more micron in the depth by tuning the pre-chirp. Other samples not a lot
>> seems to happen.
>>
>> I have spoken to Olympus about getting the 25x lens but unfortunately there
>> are two problems. 1. it is scarily expensive 2. our system would have to be
>> changed a bit to accommodate it (more expense). I would be curious to see
>> any comparisons if anyone has them, maybe it could be worth the expense:)
>> That being said though I am very, very happy with the quality we are getting
>> from the 20x lens.
>>
>> Cheers
>>
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscpy Manager
>> Central Resource for Advanced Microscopy
>> Ludwig Insttue for Cancer Research
>> PO Box 2008
>> Royal Melbourne Hospital
>> Victoria, 3050
>> AUSTRALIA
>>
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>>
>> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>>
>>
>> ________________________________
>>
>> From: Confocal Microscopy List on behalf of Adrian Smith
>> Sent: Fri 29/05/2009 5:25 AM
>>
>> To: [hidden email]
>>
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:
>>
>>> It is true that the upright microscope multiphoton system at the
>>> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
>>> lens. This was done at installation by fitting and testing with a
>>> custom set of lenses for the lens of our choice. I just want to
>>> clarify that Olympus could always optimise their system for any
>>> objective at the time of installation. I think from memory I chose
>>> the 60x WI with cover slip correction for the inverted.
>>
>>
>> So, the question still stands - has anyone directly tested the older
>> 20x dipping lens (with tuning to fill the back aperture) against the
>> new 25x? (and is allowed to share the data?)
>>
>> Apparently a theoretical question for those of with existing systems
>> but of interest none the less :)
>>
>> Steve (or Cameron) - you might also be able to comment of the
>> performance of the Olympus system with the pre-chirp on the DeepSee on/
>> off (assuming you can turn the compensation off?)
>>
>>
>> Regards,
>> Adrian Smith, Centenary Institute
>>
>>
>> --
>> Stephen H. Cody
>
>
> ---------------------------------------------------------------------
>
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
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[ezeiss]

All we need is a 45mm polorizer.
Erich
Sent from my Verizon Wireless BlackBerry

-----Original Message-----
From: "Armstrong, Brian" <[hidden email]>

Date:         Tue, 2 Jun 2009 13:33:34
To: <[hidden email]>
Subject: Re: Chirp


Thanks for the info. I figured it would be accomplished through folding. I did not think about the high index glass.
Still, I think 20cm seems like a long way to move these prisms, especially if you are doing it a lot. Is the tuning consistently repeatable with the Deep See and Coherent systems? Anyone care to comment?
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, June 02, 2009 11:40 AM
To: [hidden email]
Subject: Re: Chirp

There are two tricks; use a very high index glass in the prisms, and
fold your design with mirrors.  My design does both of these things,
although the commercial designs use even more mirror folding to
shorten the physical distance between the prisms.  Some designs only
use a single larger prism and just fold back into it repeatedly.  The
downside to folding is you have to be careful with your alignment,
although using retroreflectors rather than flat mirrors minimizes this
as they are less sensitive to misalignment.  Using high index glass
also helps considerably, although if your pulse bandwidth is too great
(greater than about 15nm) then you can start having higher-order
dispersion issues.  Our laser is an older Spectra Physics Tsunami
model; you can vary the spectral bandwidth of the pulse by manually
adjusting the laser, so we can get optimal bandwidth and dispersion
combinations with some manual tweaking of the pulse compressor and the
laser together.  It takes about 15-30 minutes to do a wavelength
re-tune with the system we've set up (shorter if we are tuning visible
wavelengths as those are easier to work with).  Dispersion adjustment
once the wavelength is set takes about 1 minute.  I can compensate for
our standard Nikon C1 microscope and 60x dipping lens with about
10-20cm of path length even with ~15nm spectral bandwidth in the
pulses with only a little residual 3rd order distortion.  The temporal
pulse width at the sample is probably around 60-70fs with best
adjustment.  I have also done some experiments with creating strong
chirps in pulses on purpose.  With about 1m of separation I can chirp
a 15nm spectrally wide pulse out to about 2ps (negative chirp).  My
compressor design can also be set to a 'neutral' position (path length
~5cm) where it has no effect (still some very small residual 3rd order
if bandwidth is high) on the pulses.  If I bring the prisms to their
minimum distance (a couple cm) I can actually get 100-150fs positively
chirped pulses out of the compressor from 60fs pulses.  Here the high
index of the glass causes enough positive chirping that it actually
overwhelms the geometric path-length adjustment that produces negative
chirp.  Fun times!  @:-)

Craig


On Tue, Jun 2, 2009 at 11:54 AM, Armstrong, Brian<[hidden email]> wrote:

> I do remember looking at your set-up. Approximately how far do you have to move your prisms (in extreme cases, say you are going from a 5x to a 20x/0.95)?
> I always thought that you had to change the inter-prism distance an appreciable amount and wonder how they (Spectra and Coherent) accomplish this is such a small box.
> Thanks in advance,
>
> Brian Armstrong PhD
> Manager, Light Microscopy Core
> Beckman Research Institute
> 1450 East Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
> http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Tuesday, June 02, 2009 10:14 AM
> To: [hidden email]
> Subject: Re: Chirp
>
> We have a similar open-prism pair setup.  It is less convenient than
> an off-the-shelf automated system but as Evangelos has found it is
> easier to deal with changing objectives and optical setups; the
> automated systems tend to use look-up tables for specific
> lenses/conditions so if you try something new you either need a way to
> create a new table entry or have some method for manually tweaking the
> nearest table setting.  I had a paper circulating on the list a while
> ago about the prism pair setup that some of you may have received.
> You can find it as part of the SPIE proceedings for Photonics West
> 2009.
>
> Craig
>
>
> On Sat, May 30, 2009 at 6:59 AM, Evangelos
> Gatzogiannis<[hidden email]> wrote:
>>    There were some good comments on chirp and pre-chirping an IR pulse for
>> multiphoton applications.  I don't have a DeepSee or Chameleon Vision, but
>> have/had older Tsunami oscillators and Coherent Mira oscillators.  In both
>> cases I've built my own pre-chirp prism pair assemblies.  There is no
>> choice, in my opinion, between no pre-chirp an chirping.   Some of the
>> objective lenses from several manufacturers such as 20x that have less glass
>> don't chirp IR pulses as much if you are operating greater than ~100fs.  You
>> must, however, compensate for system optics if you are using 60x or 100x oil
>> objectives and going through thick samples.  I have microscopes and
>> objectives from Zeiss, Olympus, and Leica and there are differences -
>> adjustments to a pre-chirp prism pair must be made daily in my lab to
>> compensate for beam pointing stability, to address the different samples my
>> users have, and to address the different objective lenses and microscopes my
>> users work on.   I like having an open accessible prism pair assembly
>> because I feed my pulses into both a Zeiss 510 Meta and an Olympus FV300
>> confocal and between objectives, scanhead optics, and samples, I find that I
>> have to adjust this assembly frequently to optimize the image on a variety
>> of samples.  Also, if damage to your sample is a serious problem, and if you
>> have significant signal - get a Pockel's cell or similar modulator.  The
>> 76Mhz repetition rate of commercial laser oscillators is like a machine-gun
>> operating away, sometimes a hundreds of kHz - 1-2 Mhz rep rate can be
>> better.  My regards to all you out there with the Mai Tai Deep See, great
>> laser, you can't do better.  The marriage between  a  Mai Tai Depp See and
>> an FV1000 is a very good one.
>>
>>
>>
>> Good day,
>>
>>
>>
>> Evangelos Gatzogiannis
>>
>> Ad
>>
>>
>>
>> From: Confocal Microscopy List [mailto:[hidden email]] On
>> Behalf Of Stephen Cody
>> Sent: Friday, May 29, 2009 10:49 PM
>> To: [hidden email]
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>>
>> G'day Adrian,
>>
>>
>>
>> As I understand it, the Olympus special 25x dipping objective for
>> multiphoton was only available in the US when we ordered and installed the
>> Ludwig system (Melbourne, Australia). I tried in vein to get the 25X
>> objective. I think it may now be available in Australia, but even so it
>> would require a refit of the beam expander to install on the Ludwig system.
>> Olympus are very precise when installing, customising and testing of their
>> beam expander. It is definitely the most labour intensive part of the
>> installation. They not only ensure that the beam fills the back focal plane
>> of the chosen objective, they measure the beam intensity profile across the
>> beam at this plane, and make adjustments to the lens set in the beam
>> expander kit to maximise performance. This data is recorded and sent back to
>> Tokyo for approval (well that's what seemed to be happening, I could be
>> mistaken). So sadly such a comparison of lenses is not feasible. If and when
>> someone has the motorised beam expander this should be a trivial test.
>>
>>
>>
>> A similar story with the DeepSee. The pre-chirp optics are always in the
>> path of the laser, there is no pre-chirp bypass. I think this is perhaps a
>> project that would be more suited to an optics lab, rather than an
>> instrument built to investigate biology. Testing the benefit of a pre-chirp
>> would take a system such as this offline for at least a couple of months and
>> would also require many, many hours of labour by the Olympus engineer to
>> reinstall, align and test a couple of times. To bypass the DeepSee pre-chirp
>> would require dismantling the the DeepSee itself and reinstalling the IR
>> laser, and then repeating the installation process again to get back to the
>> original configuration. Unfortunately not a practical thing to test. What I
>> can tell you is that it is a very simple matter to adjust the the pre-chirp
>> with your specimen to obtain the brightest signal. A poorly set pre-chirp
>> compared to an adjusted pre-chirp makes quite a difference. But without
>> knowing what the scenario would be with no pre-chirp it is very difficult to
>> give a precise answer.
>>
>>
>>
>> My gut feeling is pre-chirping is fantastic, although Cameron has certainly
>> used it much more than I, so I deffer to him in this regard. Logically, if
>> you can see a clear benefit of adjusting the pre-chirp (more fluorescence
>> and so laser power can be reduced). One would think that this must be better
>> than a system with no pre-chirp where the pulse is spread ("chirped") by the
>> optics of the microscope, and cannot be compensated. I don't want to get
>> into brand name comparisons but I think it important to to let people know
>> that (as explained to me) It seems that different microscope manufacturers
>> have markedly different amounts of chirp associated with their Optics. So
>> the decision "to Pre-Chirp or not to Pre-Chirp" is far more important
>> depending on the brand of microscope you decide to go with. As this was the
>> first installation of its kind, little was known by anyone at the time what
>> was required. It turned out that originally the DeepSee imparted too much
>> correction for our Olympus scope, and SpectraPhysics very quickly supplied a
>> "Low Dispersion" modification to the setup. After the modification we could
>> find the "sweet spot" with the DeepSee pre-chirp extremely easily.
>>
>>
>>
>> The point being with at least some other brands, pre-chirping is probably
>> much more important. I would ignore the "company speak" that maintains that
>> a similar benefit can be achieved simply by increasing IR laser power. I'm
>> not sure if the Multiphoton List is still going (I think it's membership was
>> merged with this list). However the subject "to Pre-Chirp or not to
>> Pre-Chirp" was raised by myself on that list a couple of years ago. Someone
>> replied to me (I think personally not via the list), that they demonstrated
>> a system: Without pre-chirp they observed damage to their tissue in a whole
>> mouse, above and below the plane of focus, but the fluorophore at the plane
>> of focus was not bleached. This suggested direct IR damage of the laser, but
>> that the 2P effect at the fluorophore was not damaging. When the pre-chirp
>> was installed (or perhaps it was adjusted correctly) this direct IR damage
>> was not seen, presumably lower laser power could be used, to achieve the
>> multiphoton effect. I'm sorry I can't remember who gave me this information
>> and I don't have access to my old email anymore. But a big thank you, that
>> bit of information was exactly what I needed to make a decision and allowed
>> me to cut through the "company speak".
>>
>>
>>
>> I would imagine that a collaboration with SpectraPhysics and a microscope
>> company, so that the pre-chirp could be bypassed would be the best way
>> forward to test this. Does SpectraPhysics and a microscope company want to
>> loan me a multiphoton for 12 months (or 12 years for that matter)? I have
>> not mentioned Coherent, at the time they were only offering a third party
>> pre-chirp option. However, Coherent now offer the "Chameleon Vision" with
>> integrated pre-chirp, this too should be considered in any MPE purchase
>> decision.
>>
>>
>>
>> I have no commercial affiliation with any of the companies mentioned.
>>
>>
>>
>> Cheers
>>
>> Steve
>>
>>
>>
>> Stephen H. Cody
>> Imaging Research Fellow & Manager
>> Monash Micro Imaging - AMREP
>> Monash University
>> 6 Floor Burnet Tower
>> Alfred Medical Research & Education Precinct
>> 89 Commercial Rd, Melbourne, Australia, 3004
>>
>> www.microimaging.monash.org
>> Phone (Monash):  (613) 990 30142
>> Phone (BakerIDI): (613) 8532 1580
>>
>>
>>
>> 2009/5/29 Cameron Nowell <[hidden email]>
>>
>> Hi Adrian,
>>
>> After just having a brief look I don't think the pre-chirp unit can be
>> disabled, there is no off switch and I am pretty sure if I disconnect the
>> control box all sorts of weird things will go wrong. There is already one
>> redundant component on the Maintain system that can not be removed for that
>> reason.
>>
>> I can say that if it is not set optimally there is a reduction in image
>> quality and penetration depth. Although it does seem very dependent on the
>> sample and the fluorophore. On some samples you can gain an extra 100 or
>> more micron in the depth by tuning the pre-chirp. Other samples not a lot
>> seems to happen.
>>
>> I have spoken to Olympus about getting the 25x lens but unfortunately there
>> are two problems. 1. it is scarily expensive 2. our system would have to be
>> changed a bit to accommodate it (more expense). I would be curious to see
>> any comparisons if anyone has them, maybe it could be worth the expense:)
>> That being said though I am very, very happy with the quality we are getting
>> from the 20x lens.
>>
>> Cheers
>>
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscpy Manager
>> Central Resource for Advanced Microscopy
>> Ludwig Insttue for Cancer Research
>> PO Box 2008
>> Royal Melbourne Hospital
>> Victoria, 3050
>> AUSTRALIA
>>
>> Office: [phone]
>> Mobile: [phone]
>> Fax: [phone]
>>
>> http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
>>
>>
>>________________________________
>>
>> From: Confocal Microscopy List on behalf of Adrian Smith
>> Sent: Fri 29/05/2009 5:25 AM
>>
>> To: [hidden email]
>>
>> Subject: Re: Recommendations for commercial multi-photon system purchase
>>
>>
>> On 26/05/2009, at 4:15 AM, Stephen Cody wrote:
>>
>>> It is true that the upright microscope multiphoton system at the
>>> Ludwig is tuned to fill the back aperture of the 20x 0.95na dipping
>>> lens. This was done at installation by fitting and testing with a
>>> custom set of lenses for the lens of our choice. I just want to
>>> clarify that Olympus could always optimise their system for any
>>> objective at the time of installation. I think from memory I chose
>>> the 60x WI with cover slip correction for the inverted.
>>
>>
>> So, the question still stands - has anyone directly tested the older
>> 20x dipping lens (with tuning to fill the back aperture) against the
>> new 25x? (and is allowed to share the data?)
>>
>> Apparently a theoretical question for those of with existing systems
>> but of interest none the less :)
>>
>> Steve (or Cameron) - you might also be able to comment of the
>> performance of the Olympus system with the pre-chirp on the DeepSee on/
>> off (assuming you can turn the compensation off?)
>>
>>
>> Regards,
>> Adrian Smith, Centenary Institute
>>
>>
>> --
>> Stephen H. Cody
>
>
> ---------------------------------------------------------------------
>
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
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