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  • 1.  Are White Light Lasers worth the money?

    Posted 03-07-2024 14:50
    A couple of investigators recently suggested the idea that our imaging core should purchase a white light laser for one of our core's existing confocal microscopes.  I am familiar with the standard "Pros" in support of White Light laser sources, but I am also starting to hear whispers of some serious "Cons".  I am wondering if anyone out there has actual experience with them.  If so, I am curious to know what an annual service contract costs and what considerations should be taken into account before investing in one.  If anyone knows, I would also be curious to know what the current price range is for White Light lasers.

    Anthony


    Anthony S. Stender, Ph.D. 

    Microscopy Core Manager

    Creighton University



  • 2.  RE: Are White Light Lasers worth the money?

    Posted 03-11-2024 01:14
    Edited by Mahmud Siddiqi 03-11-2024 01:25

    I have two Leica confocals with WLLs (NKT Photonics), both acquired with the systems through Leica. In my experience, there are only a few 'cons' --

    Initial costs: A WLL can add $100K - $200K to the cost of a full system, when compared to a system with fixed-wavelength diode lasers. However, this is not so different from adding a laser for multiphoton fluorescence. Both lasers are bundled into the confocal system service contract, so I am unable to give you numbers on a service agreement for the laser in isolation. 

    Repair turnaround times: In the rare instance (twice in ~9 years) that the WLL needs factory repairs, the turnaround time can be long (weeks - months). In my case, this was not overly painful, as a loaner WLL was available.

    Somewhat lower output power at selected wavelengths: This is less of an issue with newer versions. While diodes can provide more mW at their specific wavelengths, this is rarely an issue for biological specimens which can withstand only a fraction of the maximum available excitation laser power in any case. If higher powers are required, e.g. for photobleaching, applying e.g. 512, 514, 516 nm simultaneously delivers more than enough energy. 

    Tuning range: Again, less of an issue with newer versions. On my older system I have 405nm and 445nm diodes in addition to the WLL (470-670nm); on the newer one only the 405 nm diode is required (WLL 440-830nm).

    A sort of 'global con' is that the rest of the confocal system has to be able to take advantage of the various capabilities and characteristics (tunability, pulse synchronization, etc.) of the WLL to make the purchase really worth the price, especially if you will be adding only the laser. What kind of confocal would you be retrofitting?

    An argument can be made that having the ability to output 517nm instead of 514nm is a luxury (although I did have to do exactly that for a recent imaging experiment). However, if your users bring you uncharacterized fluors, or unusual fluor combinations, it's very hard to go back to fixed laser lines after using a WLL. 

    If there's a specific drawback of concern, or any specific questions,  I'll be happy to elaborate as best I can...

    Mahmud

    Mahmud Siddiqi, PhD

    Imaging Facility Manager

    Carnegie Institution / Embryology





  • 3.  RE: Are White Light Lasers worth the money?

    Posted 03-18-2024 08:35
    Thank you, Mahmud, for that perspective.  It was quite helpful.  For what it's worth, if we purchase a WLL, it would be part of a Leica system.

    My primary concerns were the cost of a service contract and laser power.  I have previously heard that the tradeoff for a WLL is power, so I'm glad you addressed that.  I'm continuing to look into the cost of a service contract, since our core needs to keep that in mind for any new purchase.  It's also helpful to hear about your experience with laser repair.  That was something I didn't think about, but it is definitely something I need to keep in mind.

    At this point, I am simply doing some fact-finding.  It is unclear if there will be enough support for a WLL, at least in the short-term.

    Anthony



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  • 4.  RE: Are White Light Lasers worth the money?

    Posted 03-19-2024 10:40

    Regarding the WLL, keep in mind it is a 80 MHz pulsed laser.  Therefore, you can do time domain FLIM or gating with excitation at any wavelength from 440 to 790 nm.

    With a detector sensitive out to 850 nm, this is also a way to add functionality for new far red probes.

    Cheers-

    Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

    NYU Langone Health, 540 First Avenue, office MSB 0657A (elevator to MSB 6 or Smilow 4)

    main lab Smilow C-17, New York, NY  10016

    Office: 646-501-0567 Cell (DO NOT TEXT): 914-309-3270  Michael.Cammer@med.nyu.edu 

    http://nyulmc.org/micros  http://microscopynotes.com/

    Scheduling the time you want is far more reliable by phone call.  Why not provide your phone number?

     

    Probably nobody reads this part, but everybody should and heed it:

    Acknowledgement in publications and presentations of Microscopy Core performed work is vital to secure support and funding necessary to maintain this valuable research resource.   For publications that include work performed in the core, please use the acknowledgement statement "We thank the NYU Langone Microscopy Core for experimental and technical support" and include required grant numbers as listed here http://microscopynotes.com/ilabnyu/acknowledgements2017.pdf
    Please also consider staff for co-authorship if they played a key role in the study. 

     

     




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  • 5.  RE: Are White Light Lasers worth the money?

    Posted 03-19-2024 12:53
    Hi Anthony (and everyone),

    I don't have any info on maintenance since our WLL system, a Lecia STELLARIS 8 with FALCON and STED, was installed just last year, but I do want to highlight that the WLL makes it super simple to increase the number of fluorophores in a single sample you can separate with "filter" like settings (instead of spectral unmixing).   

    I've had users easily image DAPI, 488nm-, 555nm-, and  594nm-excited fluors in the same sample (RNAScope) and AlexaFluor 594, Atto647N and CF680R in the same sample (those last three were all at STED resolution with only the 775 depletion laser, no less). Since I have the newer, longer range WLL I suspect we could easily do 7 channels-DAPI, 488, 555, 594, 647, 680, 760-with only modest staining optimization, and maybe even 8.   Haven't had a anyone try >5 channels or >760nm-excited fluors yet.

    Caveats: I haven't tested it with fluorophores that have substantial overlap of both excitation and emission curves--like CFP+GFP or GFP+YFP. Also, live samples that need more channels than the number of detectors on the system will be a struggle. As a real life example of what doesn't work, I have a group doing live imaging of 8 organelles that each have a different fluorophore (some FPs, some dyes) which requires an array detector with spectral unmixing to get the necessary speed--there's no time to wait for frame switching and hardware changes on my 5 detector WLL system.

    Good luck with your decision. Happy to chat directly if it would help.

    Wishing you peace and good health,
    Wendy

    -----------
    Wendy Salmon, MA  (she/her)
    Director, Hooker Imaging Core

    Dept. Cell Biology and Physiology, Univ. N. Carolina-Chapel Hill
    109 Mason Farm Rd, 236 Taylor Hall (shipping)
    CB 7545 (USPS)
    Chapel Hill, NC  27599-7545



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  • 6.  RE: Are White Light Lasers worth the money?

    Posted 03-19-2024 19:21
    Edited by Mahmud Siddiqi 03-19-2024 19:32

    Getting into Leica specifics now --

    The 'correct' pairing is the WLL with Leica's HyD detectors. If there are none on your system, then the price/performance ratio for the WLL becomes significantly less favorable. However, doing a HyD detector upgrade at the same time as adding the WLL is not so much more expensive than the laser (well, the first HyD is ~$25K, additional HyDs are $6K-ish; this pricing is from a few years ago).

    HyDs have some drawbacks, but enable a wide range of capabilities. I've been very happy with them (even upgraded an old SP5 with three). As Wendy and Michael mentioned, WLL pulse sync allows gating, FLIM, and photon counting, and more fun & games. Increased sensitivity also mitigates a lot of the laser power anxiety. 

    Re spectral unmixing -- any thoughts about WLL/HyD multiple tau gating on a single HyD? If the overlapping fluors have differing lifetimes, and are bright enough, they could be separable...

    Mahmud



    ------------------------------
    Mahmud Siddiqi, PhD
    Imaging Facility Manager
    Carnegie Institution for Science / Embryology
    Baltimore, MD USA
    ------------------------------

    Original Message


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