NetNotes
In summary, the SCH is not a tunable laser, so it’s hard to com- pare with a tunable Ti:Sapph. It might work OK in a home-built single-purpose system, but I wouldn’t bet on it if connected to a commercial point-scanning confocal microscope. No commercial interest, but I remember Torlabs’ tunable and broadband femto- second lasers were quite competitive (read: cheaper than Coherent). Zdenek Svindrych
zdedenn@gmail.com
I think it would be a hard laser to use in a general purpose
system. Te pulse length is too short which will make setting the dispersion compensation precisely important, and for best results you may actually need to correct for both GVD and third order dispersion (TOD). At the same time the power output is low, which will make efficient use of power critical, which is hard if you need to add (lossy) TOD correction. Finally, since the band- width is 200nm, all optics will have to be extremely well achro- matized (not just coated) for that (fairly unusual) wavelength. Using normal IR coated (but visible achromatized) objectives will give 950nm light focused tens or even hundreds of microns away from where 1150nm light focuses, which will not work. Similarly, the scanner optics will need to be extremely well corrected, es- pecially for lateral color. All of this is doable, but hopefully you’d be designing the microscope around that laser and not expecting the laser to work with an existing microscope. One other general question I have is the odd spectrum, presumably generated from nonlinear fiber interaction. I see a spec for long term output sta- bility (3 hr), but not one for noise. If this is a compressed nonlin- ear fiber output, the shot-to-shot spectrum can be noisy, which is fine for low-speed imaging (averaging thousands of pulses) but might be a problem if looking at resonant scanning (averaging a few pulses). Mike Giacomelli
mgiacome@ur.rochester.edu
These comments are very helpful. I would like to continue
this discussion, and to change the direction to include the cur- rent fixed line fs lasers on the market. What is the opinion on using fixed line lasers such as the Axon range from Coherent in place of something like the Incite X3 or Chameleon tunable lasers? (that is, using a 780, 920 and a 1040/1064 nm set of fixed line lasers that would cover the absorption cross-section of most common fluorophores). Again, the emphasis here is on the maintenance and lifetime of the light source. It would be great to see what those already using MP systems think about this, and their wish list for an MP system to be used in a core facility. Peter Owens
peter.owens@nuigalway.ie
Tat is something I have thought about. My experience with
1040nm Ytterbium fiber lasers has been positive. We have two from Menlo and they work extremely well. You push a button and 3 seconds later they’re mode locked at 2.5W. We have even put them in mobile cart systems, bumped them into elevators, driven them to other sites, and they still work normally. Conversely, our previous Coherent Chameleon had trouble over its entire life de- spite repeated service from Coherent. Not sure if we just got a lemon, but reliability was night and day when we moved to fiber lasers. I can’t comment specifically on the 920 systems since I’m not sure how they work internally (or if they even use the same mechanism for frequency shiſting). I think the problem with go- ing with 3 separate units is that it is going to get expensive. Te Ytterbium models are cheap, but the frequency-doubled Erbium and the frequency-shiſted 920 nm units are significantly more ex- pensive, especially the Ti:Sapph class power. Not sure what Co- herent charges these days, but you might end up spending more
60
than it would cost for a tunable Ti:Sapph and a service contract that covers swapping out the Ti:Sapph when it gets into trouble. You may also have users complaining about not being able to hit lower wavelengths (for example, 740 nm for NADH), although 780 will work in many of these applications. Mike Giacomelli
mgiacome@urrochester.edu
To echo Mike’s comments, more lasers mean more expense
and also more points of failure over their lifespan. A good single laser or integrated system with effective customer support from the manufacturer is the optimal condition. As an aside, I’ve watched the quality of some of the common brands of lasers slip over the last decade, and Mike’s experiences mirror my own in several cases. Craig Brideau
craig.brideau@
gmail.com
One notable advantage of the proposed multi-line setup is
that fast multi-color imaging (possibly line-by-line switching) is possible. Te Chameleon requires time to tune to a new wavelength (seconds), so 99% of the time people use a single wavelength for an experiment. Tis may be different for lasers with dual output, but the “free” line (typically fixed at 1045 nm) is not the most useful wavelength. I haven’t seen such a three-laser setup, but for a core facility it is important that the lasers are well integrated into a commercial microscopy system. Zdenek Svindrych
zdedenn@gmail.com
If building a microscope for one very specific application that
is known in advance, then fixed-line lasers are fine. But in a core, we never know what research needs will be required. We have dif- ferent labs that work at 920, 930, and 940 nm. Tis may not sound like a big difference, but for continuity with past experiments or for second harmonics, being able to hit these subtle differences is important. Similarly, when using around 1150 nm, there are second harmonics that we can clip from the standard red filter set by tuning down or up a little. And we have customers, includ- ing chemists developing new probes, who want a wide variety of wavelengths for various reasons. I’m not an expert on the lasers but am expert on providing services across the spectrum. Which gets to a point about needing two wavelengths. If an investigator cannot wait 5 sec between fields while a laser retunes (and the soſtware needs to be able to handle the pause), then two lasers are needed. Also, changing wavelengths may require refocusing col- limators, other optical elements, or the objective itself to assure that the wavelengths are focused on the same plane in the sample. For live cell work, we like to have the lasers alternate for each line that is scanned (and some people might want the speed of simul- taneous lasers, but then we get into a discussion of higher pho- ton flux per time). We don’t worry about the laser pulses being in synch, but for some applications this may also be important. Michael Cammer
michael.cammer@med
.nyu.edu
Non-Fluorescent Bright Field Confocal Listserver Risking a slight departure from the confocal microscopy topic;
Do you know of any compounds that I may use as a non-fluorescent dye to increase the contrast in brightfield mode? In this case, specifi- cally of PFA-fixed mouse brain slices? Te goal is to make them more clearly visible on a brightfield system, while at the same time avoiding the introduction of fluorescence to keep the full spectrum available for other fluorescent markers. For example, hematoxylin/eosin and cresyl violet are common brightfield stains, but they introduce fluorescence. Tanks in advance! Jelle Postma
j.postma@
science.ru.nl
www.microscopy-today.com • 2022 July
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