Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/laser-choices-for-multiphoton-tp7585404p7585407.html
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Michael,
you probably are aware that some red dyes do just nicely when excited
around 860 or so, although is most likely not the S1-state (Mütze et
al., Excitation Spectra and Brightness Optimization of Two-Photon
Excited Probes. doi:10.1016/j.bpj.2011.12.056)
Having said that, I like the possibility to tune up to 1300 nm. It gives
you much more flexibility. e.g. exciting Eosin is not a problem, or the
red FPs. Also I like THG a lot, usually around 1275 nm excitation, with
the THG signal at 425 nm and SHG at 638.
So far we didn't have an application where we needed a high laser line
(e.g. 1200) and a low one (850) simultaneously. If one comes around we
would have to tune the laser, which takes time. If this is a frequent
request at your site, you might be better of with two lasers. If money
were no issue, I'd suggest two lasers tunable up to 1300nm...
Our old LaVision BioTech and also our new Leica SP8 both support
sequential scanning with several wavelengths (tuning in-between), I
suppose the others do too.
I believe the fixed 1046 is useful only if you want to do CARS. Or if
you happen to have fluors that are nicely excited at that range. But I
don't know any. In that respect a TiSa-OPO combination may be more
useful were you can take out 10% of a 4 W laser to image directly
(e.g.834 nm) and the 90% to generate a long wavelength.
I wouldn't worry too much about cooking. One, the absorption of tissue
is very low between 1200 and 1300. Two, the transmittance of the
microscopes is very low, so you might get not so much out of the
objective (still plenty to do fluorescence). Three, if you are still
worried, a resonant scanner might be a good idea.
Steffen
Am 07.07.2016 um 18:54 schrieb Cammer, Michael:
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>
> We are considering a multiphoton purchase and a big issue is illumination. Putting budget constraints aside and thinking purely of imaging non-destructively in live tissues, what would give us the most flexibility for exciting multiple probes for maximum signal while minimally damaging the biology?
>
>
>
> We are used to using lasers that tune from approx 700 to 1060 nm and mostly use 890 to 930 nm, but this does not provide good red imaging. If money were no issue, would we be wise to get a laser such as the dual line Insight or two lasers from 690 to 1080 nm?
>
>
>
> Some of the questions that have come up are:
>
> * We think a laser that tunesup to 1300 nm would solve the red imaging problem, but for bluer probes (CFP, GFP, etc) & second harmonics of collagen would we need to use a different wavelength? If so, how long does this take and do the commercial systems support this?
>
> * With a dual line laser that tunes out to 1300 nm is the fixed 1046 nm line really useful?
>
> * Does a dual line system cook the sample?
>
>
>
> Any thoughts on this (and on specific multiphoton scopes) greatly appreciated.
>
>
>
> Thank you!
>
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
> Cell: 914-309-3270 Office: Skirball 2nd Floor main office
>
http://ocs.med.nyu.edu/microscopy &
http://microscopynotes.com/>
>
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging
Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany
http://www.bioimaging.bmc.med.uni-muenchen.de