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 <[log in to unmask]>
To: [log in to unmask]
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 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 to20their 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 <[log in to unmask]>


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: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104

ht
tp://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: [log in to unmask]

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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