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<[log in to unmask]> 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:[log in to unmask]] On
> Behalf Of Stephen Cody
> Sent: Friday, May 29, 2009 10:49 PM
> To: [log in to unmask]
> 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 <[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
>
> 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: [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
|
