Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi,
In the Zeiss the problem of removing the AOM is that it has a mechanical
shutter that opens before the galvos start to scan. This means that you
will get a burned spot at the center of the FOV (I tried that one). You
can build some electronics to synchronize the slow motor/bar based
shutter with the AOM signals but I would believe it will be an pain...or
maybe not, I have to check that!
Talking about pre-chirping do you know someone using something similar
to maitai deepsee in a Coherent Mira laser?
Regards,
NM
Craig Brideau wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Our own lab has found that modifying confocal microscopes for 2-photon
> is a tricky business. As mentioned before, an AOM will cause problems
> with the laser. If there is any way to bypass the AOM in your system,
> or simply remove it, this will help.
> Another issue is optics and objectives. Most optics and objectives
> are designed for the visible range rather than the NIR. From my own
> experience certain medium-high-end Nikon and Olympus lenses tend to
> have the best NIR performance, and most others are terrible; absorbing
> 90% or more of the light. A crude test is to use a laser power meter
> to measure the power after your objective. Place the detector head
> some distance away where the light is diverging but still all of it
> hits the detector surface; the focal point can damage your detector.
> Compare this to the power before the microscope to get your system
> losses. Don't be afraid to ask your manufacturer for the NIR
> transmission spectrum for your lenses.
>
> Craig
>
>
> On Dec 7, 2007 5:06 AM, "José A. Feijó" <[log in to unmask]> wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> thanks George, it's good to hear about good experiences (and definitely
>> you raise out some good points). But again 10x and 770nm may not be what
>> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
>> "the" perfect 2P dyes!
>>
>> George McNamara escreveu:
>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Hi Jose,
>>>
>>> Thanks for the great post!
>>>
>>> On City of Hope's multiphoton rig, with a Chameleon split between two
>>> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
>>> hundreds of um into freshly excised mouse brain stained with Hoechst
>>> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
>>> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
>>> dilution from 10 mg/mL bottle into dH2O), then can dilute into
>>> whatever the brain is in. 770 nm is also optimal for several (not
>>> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
>>> Alexa Fluor 488 tomato lectin could be used to label blood vessels
>>> immediately before sacrificing the mouse.
>>>
>>> Edrun - This was with fairly low power, but don't be afraid to crank
>>> up the power! Also, try more than one objective lens, and test
>>> different wavelengths.
>>>
>>> If working with fixed tissue: If you need to make a fixed specimen
>>> transparent, there are several recipes for clearing tissue - see
>>> Robert Zucker's papers using BABB for example. An interesting mounting
>>> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
>>>
>>> best wishes,
>>>
>>>
>>> George
>>>
>>>
>>>
>>> At 06:28 AM 12/7/2007, you wrote:
>>>> Search the CONFOCAL archive at
>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>> the problem I believe is not 2P physics, but the Zeiss 510. We've
>>>> been trying to keep a low profile about this, but it's certainly
>>>> frustrating that we can't really get much out of 510 under 2p, and
>>>> it's painful that we still get much better results with a
>>>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
>>>> of design and technology (do mind, sharing the same laser and optical
>>>> path, we just diverge form one to the other with a simmple
>>>> reflector!!). During the purchase process I made a number of
>>>> inquiries about a number of physical parameters affecting the
>>>> negative GVD performance of the system, namely the AOM and the
>>>> external detectors path, and NEVER got any satisfactory, even less
>>>> quantitative answer from anyone from Zeiss. The AOM sucks, it
>>>> destroys the power and the pulsewidth, one good reason for you to see
>>>> such small difference, penetration is pretty much dependent on both
>>>> of these. We've tried a simple hand operated ND filter, but
>>>> unfortunately the fly-back of the beam in the 510 turned out to be
>>>> nasty for most samples. The external PMT's optical design sucks^2,
>>>> and does not help either.
>>>>
>>>> In our hands, and after 2 years of fighting the beast and trying to
>>>> have some answers from Zeiss, we are changing our initial plan of
>>>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
>>>> 2P to make use of all the nice software features and (oh insane
>>>> naivety!) eventually the META detector, and now we find ourselves
>>>> buying video amplifiers from China to repair the Bio-Rad amplifiers
>>>> and try to keep it running, and eventually downgrade the Zeiss to
>>>> bare confocal (eventually we will have tow external for sale, in case
>>>> anyone's interested...).
>>>>
>>>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
>>>> Zeiss people could eventually learn from their 2P design investment,
>>>> and their user database experience. That is surely not the case with
>>>> the 510. In short I fail to meet anyone with a different experience
>>>> than ours, if there is someone out there on the list that actually
>>>> managed to bend a 510 to fulfill the kind of expectations we've got
>>>> used to with other machines, and have been reported in many papers
>>>> out there, we would be very interested in learning how. By now I
>>>> have problems seeing it coming from Zeiss, they are probably more
>>>> concerned on developing the forthcoming 610 or whatever (and then
>>>> perhaps propose costly upgrades "true that one didn't work, but this
>>>> one will..."), but the sad reality is that I feel that Zeiss has NOT
>>>> come of age in matters of 2P. Not even with the monopoly of the patent.
>>>>
>>>> I would be very happy to retract myself to the list if I'm proven
>>>> wrong in this regard...
>>>>
>>>> Jose
>>>>
>>>> Edrun Andrea Schnell escreveu:
>>>>> Search the CONFOCAL archive at
>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>> Dear list,
>>>>>
>>>>> it is a known fact that a two photon laser will penetrate deeper into
>>>>> tissue, and we have done several experiments to show this, but without
>>>>> success. We have used spheroids labeled with several dyes, and imaged
>>>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
>>>>> 2) two
>>>>> photon laser and descanned detector and 3) two photon laser and
>>>>> non-descanned detector. We have only found a difference of about
>>>>> 10-20 um
>>>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
>>>>>
>>>>> So I'm wondering if anyone else have done this kind of experiment
>>>>> and have
>>>>> any tips as to how to image this increase in penetration depth? Type of
>>>>> sample and dye etc.?
>>>>>
>>>>> Thanks a lot!
>>>>>
>>>>> Regards,
>>>>> Edrun A. Schnell
>>>>>
>>>>> --
>>>>>
>>>>> Edrun Andrea Schnell
>>>>> Divisional engineer,
>>>>> Dept. of Physics, NTNU
>>>>> Hogskoleringen 5, 7491 Trondheim
>>>>> Norway
>>>>>
>>>>>
>>>> --
>>>>
>>>>
>>>> **********************************************************
>>>> Jose' A. Feijo', Prof.
>>>> ----------------------------------------------------------
>>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>>>> PT-1749-016 Lisboa, PORTUGAL
>>>>
>>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>>>
>>>> and/ e
>>>>
>>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>>>
>>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>>>> __________________________________________________________
>>>> e.mail: [log in to unmask]
>>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>>>> **********************************************************
>>>
>>>
>>>
>>>
>>>
>>> George McNamara, Ph.D.
>>> University of Miami, Miller School of Medicine
>>> Image Core
>>> Miami, FL 33010
>>> [log in to unmask]
>>> [log in to unmask]
>>> 305-243-8436 office
>>> http://home.earthlink.net/~pubspectra/
>>> http://home.earthlink.net/~geomcnamara/
>>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>> Analytical Imaging Core Facility)
>>>
>> --
>>
>>
>>
>> **********************************************************
>> Jose' A. Feijo', Prof.
>> ----------------------------------------------------------
>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>> PT-1749-016 Lisboa, PORTUGAL
>>
>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>
>> and/ e
>>
>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>
>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>> __________________________________________________________
>> e.mail: [log in to unmask]
>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>> **********************************************************
>>
>
--
Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
http://uic.igc.gulbekian.pt
http://www.igc.gulbekian.pt
phone +351 214464606
fax +351 214407970
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