NetNotes
age where it needs to be replaced. I usually try to purchase a year in advance as to when I think the confocal instrument will become outdated. Our instruments are also heavily used and I do not want to take the chance of being down one instrument. Brian Armstrong
barmstrong@coh.org
White Light Lasers Confocal Microscopy Listserver Do any companies other than Leica provide white light lasers (WLL)
with their confocals? Or if any other technology can replicate the free selec- tion of wavelength laser light? On the other hand, what are the disadvan- tages of the WLL? Konstantín Levitskiy
microscopia-ibis@us.es
I am not aware of any company other than Leica offering a WLL
on a confocal microscope (LaVision/Miltenyi offers it on their light sheet systems), but I may well be wrong. Te WLL is fantastic as it is very flexible, allows long wavelengths (670 nm, unless that has changed now), and also enables time gating in conjunction with the HyD detectors. It does have the disadvantage of not being so powerful at a particular selected wavelength. For imaging we rarely find that to be an issue as you can always activate two lines close together to increase the power, but it is a problem if you want to do FRAP. One other potential disadvantage is that if the WLL goes down, you have lost all of your lasers in effect, but our experience has been that Leica is pretty efficient at replacing it, though obviously service reliability varies hugely with geographical location. Alison J. North northa@
mail.rockefeller.edu
I am also very interested in the advantages and disadvantages of
the WLL. Our systems all have 4–5 lasers that cover the visible spec- trum well (405–635 nm). Given that most fluorophores don’t have a very narrow absorption spectrum, how important is the flexibility of the WLL? I think it would be very useful perhaps for spectral unmix- ing, but how oſten do users change the excitation when using the WLL? Te only laser line that I miss having now and which would be great to have on any new system is a UV. We recently decommissioned our last confocal which had the water-cooled argon with 351/364nm. I was really sad to see it go. Jacqueline Ross
jacqui.ross@
auckland.ac.nz
Te white light laser (WLL) is a continuum-based fiber laser.
Tese mostly work, but if anything goes wrong they are not easily repairable. Te power of WLLs shorter than ∼480nm is also very low, and most are unusable shorter than 450nm, so a separate 405 or 440nm laser may be needed. Te tuning system relies on acous- tic modulation or liquid crystal filtering, which works fairly well, but may need periodic checkups for tuning frequencies in the case of the acousto-optical modulator (AOM) or “twist” in the case of the liquid crystal (LC) for each wavelength band. I would recommend keeping the service contract up-to-date if just for peace of mind. Te bene- fit of such a laser is any wavelength from cyan to deep red is avail- able and even into the NIR for some systems. It is also possible to adjust the band of some of the filters to narrow or widen the spec- tral content. Wider filtering will allow for more power for improved signal. For core facilities the WLL provides a great deal of flexibility in terms of what fluorophores can be addressed, which is handy if there are a wide variety of users. It is also beneficial for users tag- ging a single sample with a large number of fluorophores as access to many spectral lines allows investigators to be very strategic with the colors they select. However, for a researcher purchasing a system for their own project, with known fixed fluorophores, the WLL is prob- ably not necessary. Te only alternative technology I am aware of are some of the semi-tunable gas and solid-state lasers, but I know of no systems that are commonly available. In the past I have considered
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www.microscopy-today.com • 2020 March
a frequency-doubled continuous-wave Ti:Sapphire laser, which can access green through the NIR, or the very rare tunable He:Ne laser, which can access numerous discrete lines from green to NIR. Craig Brideau
craig.brideau@
gmail.com
Apart from things that were already mentioned by others, the
fact that WLLs are pulsed is an advantage and a disadvantage. Gated detection with the HyD detectors is an extremely cool feature to get rid of reflected background noise. Plant people also seem to love this since you can gate out the very short lifetime chlorophyll fluores- cence. On the downside, you can excite any fluorochrome only every 12.5 seconds, so you may run into saturation faster than with a con- tinuous wave (cw) laser. Another downside is that the excitation line is not as narrow as with single lasers. In other words, it seems that the AOTF is not 100% tight against neighboring lines. In spectral detection we advise our users to keep 10 nm between excitation and detection. Tis is a very cautious approach, and if you are careful and know what you are doing you can get closer. But as a general rule we’d prefer to be on the safe side for the HyDs. Now you could argue that the gated detection reflection suppression is a problem that you wouldn’t have without the WLL, but I don’t think so. Some samples create a strong reflection near the coverslip that I don’t think is com- ing from the WLL, I know this pattern also from single line systems. Also, the option of exciting with 670 opens up another channel above 630 excitation that you don’t reach with a 488/552/594/633 battery of lasers. Maybe in some experiments it is helpful to know that there will be zero misalignment between the laser lines (except for the 405 and the WLL). Steffen Dietzel
lists@dietzellab.de
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