SPEKcheck fluorescence microscopy spectral visualisation.
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Ian Dobbie |
SPEKcheck fluorescence microscopy spectral visualisation.
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Everyone, Sorry for the outright self promotion, however we have developed a tool that we find extremely useful and I hope that others will too. SPEKcheck is a browser based tool that allows visualisation of the light paths of fluorescence microscopes as well as calculation of excitation and emission efficiencies and brightness. We have just published a paper on it at https://wellcomeopenresearch.org/articles/3-92/v1 or just jump straight to our copy at https://www.micron.ox.ac.uk/software/spekcheck/ The code is open source and freely available at https://github.com/MicronOxford/SpekCheck and can easily be run locally with lab or facility specific configuration. Please have a look and see what you think. Thanks, Ian |
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George McNamara |
Re: SPEKcheck fluorescence microscopy spectral visualisation.
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Ian, I suggest you incorporate in your 'master' dataset ALL the fluorophore and filters spectra in PubSpectra ... xlsx file (a type of XML file) inside the zip at https://works.bepress.com/gmcnamara/9 (it is open access, no idea why there is an s in https). Your list of existing web sites is minimal -- and gives no credit to Semrock Searchlight already having calculations, see bottom of their site -- part of research is scholarship, including "giving credit where credit is due" to your predecessors). You can redirect readers to our open access article (and don't forget the supplement please!) at https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cphg.42 aka https://doi.org/10.1002/cphg.42 Table 4.Useful Light Microscopy Web Sites ... section */Fluorescence spectra data and graphing sites/* (section is reproduced in p.s. below) I also want to give a shout of to Jonathan Lindsey, NCSU (and NIRvana Sciences), whose original PhotoChemCAD was a major source of PubSpectra data. They recently published 2 papers and released PhotoChemCAD 3.0, see http://photochemcad.com *** Basing brightness on "Alexa-488" is a horrible mistake on your part. I recommend fluorophore intrinsic brightness (no referencing to a 20 year old fluorophore ... at least you did not reference to 100+ year old dye): B = Extinction coefficient * quantum yield / 1000 which for Brilliant Violet 421 is: B = 2,500,000 * 0.6 / 1000 = 1,500 * I thought "B" was published by Roger Tsien, Lauren Ernst and Alan Waggoner in Pawley 2006 Handbook, https://link.springer.com/chapter/10.1007/978-0-387-45524-2_16 just skimmed over the chapter and did not see it. Maybe was in predecessor 1995 handbook chapter? I strongly encourage you to look it up change your Brightness for "version 2". I also encourage you to use the correct name of each fluorophore, for example "Alexa-488" is really Alexa Fluor 488 (ideally would also have a TM symbol, though that is probably more pain and illegibility than desirable for a web drop down list). *** Giving equal weight to Excitation and Emission in 2018 is another mistake. On both our Leica SP8 and brand new Olympus FV3000RS (thanks again to NIH S10 study section and council members, and especially U.S. taxpayers for the grant money, and Olympus for the generous trade-in credit for our Zeiss LSM510META scanhead ... I hope someday to see a photo of 'Mount Scanhead' in the backyard of Olympus H.Q.), we are using 1% laser power (i.e. Coherent OBIS 488 laser). Specifically: * fluorophore extinction coefficient not perfectly matched to laser line doesn't really matter. * Collecting as much fluorescence emission without illumination bleedthrough and/or other fluorophores bleeedthrough (including autofluorophores) is key (along with spectral unmixing. *** Single fluorophore focus in "version 1": * bleedthrough, and whether can cleanly separate with acquisition settings and/or spectral unmixing, is key. * no love for FRET or QRET (or BRET). ... There is a FRET calculator Excel file inside the PubSpectra zip download. *** 300 ... 800 nm limited range ... I suggest flexibility to 200 ... 1800 nm ( https://www.chroma.com/spectra-viewer goes 200 to 2050 nm, partly because they have additional markets). Sure, not much going on at 200-250 nm, but 250-320 nm has excitation of various autofluorophores, including several amino acids (trp, phe, tyr, maybe his). BD Biosciences is likely to have within 3 years Brilliant's that excite best at 320 nm, which I'll guess will be "Brilliant Deep UV's" (my source: Bob Balderas, BD, 3/2018 seminar at JHU ... to help get to 50plex flow). Your version 1 admits multiphoton would be good: Xhris Xu and others are now publishing advantages of ~1700 nm for 3p excitation (could also be handy for 4p), and Ralph Weissleder et al (PubMed 29410158) has pointed out advantages of 4p > 3p > 2p > 1p for fluorescence anisotropy, which is becoming more useful, re: FLARE biosensors (PubMed 29968564). Detectors list: I strongly encourage YOU to host a master data list of 'generic' detectors, such as S20 (and maybe some other 'classic' PMTs), GaAsP, GaAs detectors (PMTs, Hybrids), APDs, various front and back illuminated CCD and sCMOS sensors. *** You mention lack of photobleaching data. This is appropriate since very specific to conditions. You are lifetime-less: very practical, though becoming more important now that "fast lifetime" confocals (and some widefield cameras too) are becoming commercially available. * disclosure: I am scheduled to host a Leica FALCON demo "early Sept" (talk on afternoon Mon Sept 10 - anyone interested in hands on time can contact Leica reps), and may have more fast FLIM events here ... and am hoping to attend the TCSPC workshop in Bethesda Oct 9-10, https://www.eventbrite.com/e/12th-annual-workshop-on-advanced-tcspc-techniques-in-the-biomedical-sciences-tickets-46820517428 (would be attendee, I am not an organizer). *** I have a microscope core to manage - doubt that I want to deal with an 'instance' for each acquisition PC (or deal with getting it to work on each of 100+ potential local core users). I suggest YOU hosting everyone at your site, and make it easy to share configurations and spectra. sincerely, George p.s. Fluorescence Spectra web sites as of whenever I finished the manuscript https://doi.org/10.1002/cphg.42 Table 4.Useful Light Microscopy Web Sites ... section */Fluorescence spectra data and graphing sites/* AAT Bioquest https://www.aatbio.com/spectrum BD Biosciences: Spectrum viewer, Brilliant fluorophores * http://www.bdbiosciences.com/us/s/spectrumviewer * http://www.bdbiosciences.com/us/applications/s/spectrumguidepage BioLegend: Spectra analyzer, Brilliant Violet fluorophores * http://www.biolegend.com/spectraanalyzer * http://www.biolegend.com/brilliantviolet Chroma Technology https://www.chroma.com/spectra‐viewer <https://www.chroma.com/spectra-viewer> eBioscience (Affymetrix, Thermo Fisher Scientific), fluorPlan: Multi‐laser spectra viewer http://www.ebioscience.com/resources/fluorplan‐spectra‐viewer.htm <http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm>Spectra Viewer (requires Java) Evrogen: Spectrum Viewer http://evrogen.com/spectra‐viewer/flash/viewer.html <http://evrogen.com/spectra-viewer/flash/viewer.html> Fluorophores.org: Fluorescent Substances http://www.fluorophores.tugraz.at/substance I Love GFP https://sites.google.com/site/ilovegfp/Home/fps Lambert and Thorn, UC, San Francisco, fluorescent protein properties http://nic.ucsf.edu/FPvisualization/ Leica Microsystem: “FluoScout” http://www.leica‐microsystems.com/fluoscout <http://www.leica-microsystems.com/fluoscout> Molecular Probes/Invitrogen/Thermo Fisher Scientific: Fluorescence SpectraViewer * http://www.thermofisher.com/us/en/home/life‐science/cell‐analysis/labeling‐chemistry/fluorescence‐spectraviewer.html <http://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html> * https://itunes.apple.com/us/app/fluorescence‐spectraviewer/id421031826 (iTunes App) <https://itunes.apple.com/us/app/fluorescence-spectraviewer/id421031826%20%28iTunes%20App%29> Nightsea: Compendium of spectra web sites http://www.nightsea.com/sfa‐sharing/fluorescence‐spectra‐viewers <http://www.nightsea.com/sfa-sharing/fluorescence-spectra-viewers> Omega Optical “Curvomatric” https://www.omegafilters.com/curvomatic/ PhotoChemCAD 2.0 (Jonathan S. Lindsey) dye spectra database and chemistry calculator, NIRvana Sciences * http://www.photochemcad.com <http://www.photochemcad.com/> * http://nirvanasciences.com/?page_id=3088 <http://nirvanasciences.com/?page_id-3088> PubSpectra: Excel file data download https://works.bepress.com/gmcnamara/9 UC, San Francisco (Lambert & Thorn,2016 <https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cphg.42#cphg0404-bib-0142>): Fluorescent protein properties http://nic.ucsf.edu/FPvisualization University of Arizona: Fluorescent Spectra Database http://www.spectra.arizona.edu <http://www.spectra.arizona.edu/> Semrock: Searchlight https://searchlight.semrock.com <https://searchlight.semrock.com/> Spectroscopy Ninja: Links to spectral and fluorescence data, Spekwin32 spectroscopy software * http://www.effemm2.de/info/info_specdata.html * http://www.effemm2.de/index.html Zeiss: Interactive fluorescence dye and filter database, overview dyes, overview filter sets * https://www.micro‐shop.zeiss.com/?s=50479761ad21e5&l=en&p=us&f=f&a=i <https://www.micro-shop.zeiss.com/?s-50479761ad21e5&l-en&p-us&f-f&a-i> * https://www.micro‐shop.zeiss.com/?s=55721483f91174&l=en&p=us&f=f&a=d <https://www.micro-shop.zeiss.com/?s-55721483f91174&l-en&p-us&f-f&a-d> * https://www.micro‐shop.zeiss.com/?s=55721483f91174&l=en&p=us&f=f&a=f <https://www.micro-shop.zeiss.com/?s-55721483f91174&l-en&p-us&f-f&a-f> p.p.s. I declare no competing financial interests. The three spectral patents I am co-inventor on have all expired, and during their lifetimes were royalty-free with respect to my bank account. On 8/17/2018 1:23 PM, Ian Dobbie wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Everyone, > > Sorry for the outright self promotion, however we have developed a tool > that we find extremely useful and I hope that others will too. > > SPEKcheck is a browser based tool that allows visualisation of the light > paths of fluorescence microscopes as well as calculation of excitation > and emission efficiencies and brightness. We have just published a paper > on it at https://wellcomeopenresearch.org/articles/3-92/v1 or just jump > straight to our copy at https://www.micron.ox.ac.uk/software/spekcheck/ > > The code is open source and freely available at > https://github.com/MicronOxford/SpekCheck and can easily be run locally > with lab or facility specific configuration. > > Please have a look and see what you think. > > Thanks, > > Ian -- George McNamara, PhD Baltimore, MD 21231 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome) http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 http://confocal.jhu.edu July 2017 Current Protocols article, open access: UNIT 4.4 Microscopy and Image Analysis http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract supporting materials direct link is http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 figures at http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures |
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Jennifer Waters |
Re: SPEKcheck fluorescence microscopy spectral visualisation.
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In reply to this post by Ian Dobbie
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** George, Ian has clearly put a significant amount of time and effort into building a tool for our community. I suspect you don’t care since so many of your posts have the same tone – but IMHO your post here is unappreciative, overly critical and unnecessarily harsh. You should hope that no one puts as critical eye on your post - given the errors and self-promotion throughout. Jennifer |
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Talley Lambert |
Re: SPEKcheck fluorescence microscopy spectral visualisation.
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In reply to this post by Ian Dobbie
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** George, this response is an embarrassment to the community. I'm not sure if you imagine that you're being "constructively critical", but it comes off as self-centered, thankless, and, in places, ignorant and short-sited. Granted, all projects have room for improvement (and this *should* be a forum to discuss those suggestions), but to state that every design decision you disagree with was a "mistake" (even a "horrible mistake") without anywhere acknowledging the tremendous amount of effort that went into this very useful (and free!) resource, is rude and puts quite a bit of weight onto your own opinion (which, in my opinion, is starting to feel extremely dated and tiring). It would be one thing if this were a one-time offense, but you have a long track record here of hijacking threads into diatribes of thinly veiled self-promotion, advertisements for pub spectra, and reminders that you have have an image somewhere on the microvolution website. In this particular thread about SPEKcheck, we don't need to know the percent laser power you use on your confocal, that you'd like to thank the US taxpayers for your S10 grant, that you're hosting a Leica FLIM demo this September, that you have a FRET calculator excel file, or your long personal list of spectra information URLs which this list has seen many times. Ian, thanks a lot for this awesome tool. As usual from your group, the codebase is super clean and well thought out, and the tool helps turn large spreadsheets of spectra spreadsheets into actually useful and actionable information that many microscopists would benefit from. While George may struggle to appreciate the portability of your design, I think the fully client-side nature of the code is pretty cool and will certainly be useful for those who want to modify or extend the functionality on their own. George, if you're looking for a similar microscope-specific spectra tool where you don’t need to run your own instance, you can check out https://www.fpbase.org/microscopes, which stores the optical configs and spectral info in the server-side database. Thanks again Ian, and congrats on the release. PS. George the "s" in https just means the webpage uses an encrypted communication protocol, and is not related to or incompatible with open-access. |
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Alison J. North |
Re: SPEKcheck fluorescence microscopy spectral visualisation.
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