Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/SPEKcheck-fluorescence-microscopy-spectral-visualisation-tp7588555p7588565.html
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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.42aka
https://doi.org/10.1002/cphg.42Table 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.42Table 4.Useful Light Microscopy Web Sites ... section */Fluorescence
spectra data and graphing sites/*
AAT Bioquest
https://www.aatbio.com/spectrumBD Biosciences: Spectrum viewer, Brilliant fluorophores
*
http://www.bdbiosciences.com/us/s/spectrumviewer *
http://www.bdbiosciences.com/us/applications/s/spectrumguidepageBioLegend: Spectra analyzer, Brilliant Violet fluorophores
*
http://www.biolegend.com/spectraanalyzer *
http://www.biolegend.com/brilliantvioletChroma 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/substanceI Love GFP
https://sites.google.com/site/ilovegfp/Home/fpsLambert 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/9UC, 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/FPvisualizationUniversity 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.htmlZeiss: 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:
--
George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamarahttps://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/44962650http://confocal.jhu.eduJuly 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstractsupporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures