Survey on Advanced Fluorescence Microscopy

[Olaf Selchow]

*****
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.
*****

Dear List, dear Microscopists, Biologists, Scientists,

I work as a freelance Microscopy & BioImaging Consultant in Germany and I would like to ask you for the favor to participate in a short survey on Advanced Microscopy Techniques that I have put together on google.

It is about if you use FLIM, FCS, Single Molecule Imaging etc, and what you think is needed to make these technologies more accessible to a wider part of the life sciences. I aim to figure out trends and needs.

The survey is divided in 4 sections and will take you about 10 to 12 minutes

========
Please find the survey following the link          https://goo.gl/forms/b7J32fAgqvvBaw452
========

The purpose of this project is to optimise my consulting input for microscopy developing companies, with the goal to improve the available hardware and make the above methods accessible to a wider user base. It is not a purely commercial project because the insights I gain will be beneficial to both, the industrial partners I work with and the academic microscopy community.
This survey is targeted to microscopists, biologists, life scientists, medical doctors and microscopy-users in the life science field in general.

The survey is anonymous. Be assured that no personal data will be collected. The results will not be published or used otherwise than indicated. Please fill the form only once unless you have multiple professional roles, like, for example, running a microscopy core facility and, in a different role, carrying out research as a postdoc or group leader.

If you are interested in discussing results with me, you are invited to contact me at [hidden email] or via Linkedin (www.linkedin.com/in/selchow) after June 1, 2018 (It will take a while to sort the data ...)

Thank you very much in advance for your time and input.

With best regards,
Olaf

____________________________
Dr. Olaf Selchow
Microscopy & BioImaging Consulting
[hidden email]
[phone]
skype: olaf.selchow

[Ralf Palmisano]

*****
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.
*****

Dear Mods,

correct me if I am wrong. Did we not agree to mark commercial postings
as commercial?

Best
Ralf

Am 11.04.2018 um 12:04 schrieb Olaf Selchow:

> *****
> 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.
> *****
>
> Dear List, dear Microscopists, Biologists, Scientists,
>
> I work as a freelance Microscopy & BioImaging Consultant in Germany and I would like to ask you for the favor to participate in a short survey on Advanced Microscopy Techniques that I have put together on google.
>
> It is about if you use FLIM, FCS, Single Molecule Imaging etc, and what you think is needed to make these technologies more accessible to a wider part of the life sciences. I aim to figure out trends and needs.
>
> The survey is divided in 4 sections and will take you about 10 to 12 minutes
>
> ========
> Please find the survey following the link          https://goo.gl/forms/b7J32fAgqvvBaw452
> ========
>
> The purpose of this project is to optimise my consulting input for microscopy developing companies, with the goal to improve the available hardware and make the above methods accessible to a wider user base. It is not a purely commercial project because the insights I gain will be beneficial to both, the industrial partners I work with and the academic microscopy community.
> This survey is targeted to microscopists, biologists, life scientists, medical doctors and microscopy-users in the life science field in general.
>
> The survey is anonymous. Be assured that no personal data will be collected. The results will not be published or used otherwise than indicated. Please fill the form only once unless you have multiple professional roles, like, for example, running a microscopy core facility and, in a different role, carrying out research as a postdoc or group leader.
>
> If you are interested in discussing results with me, you are invited to contact me at [hidden email] or via Linkedin (www.linkedin.com/in/selchow) after June 1, 2018 (It will take a while to sort the data ...)
>
> Thank you very much in advance for your time and input.
>
> With best regards,
> Olaf
>
> ____________________________
> Dr. Olaf Selchow
> Microscopy & BioImaging Consulting
> [hidden email]
> [phone]
> skype: olaf.selchow

--
Ralf Palmisano
Head - Optical Imaging Centre Erlangen

Fellow Royal Microscopical Society
Member Royal Society of Medicine

Speaker Scientific Advisory Board "German Society for Microscopy and Image" Analysis
Board of Directors Core Technologies for Life Science

Hartmannstr. 14
91052 Erlangen, Germany

[phone] (Office)
[phone] (Secretary)
[phone] (Fax)

www.oice.uni-erlangen.de

[Olaf Selchow]

*****
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.
*****

*** Please note that this is a commercial posting - or maybe a kind of "semi-commercial" posting ;-) ***

Dear Ralf, dear fellow listers,

Please apologize that I didn't mark my earlier posting appropriately. This was a mistake that should not have happened.

Still, please let me explain that I would like to ask you all for the favor to support my information gathering with your feedback in the below survey on Advanced Microscopy Techniques (FLIM, FCS, SMD etc).

Yes, I am making my living as a professional microscopist ... and will use the information for my professional activities - as probably most of you do in one or the other way. And yes, I agree that I should have made this more transparent. On the other hand, be assured that I am not intending to sell you anything. If I am doing my job right, I am advocating your interests and requirements with commercial manufacturers and suppliers of microscopy equipment and software.

I have been asked by some of you about "what would be in for you" with this survey. Well, I am a one-man show and I don't have the ressources to incentivize participants with anything else but the promise that I will use the feedback to advocate your requirements with those companies that I have the chance to work with.

Please, again, accept my apologies for not making the nature of my posting very clear in the beginning.
With best regards,
Olaf


========
Please find the survey following the link          https://goo.gl/forms/b7J32fAgqvvBaw452
========

Thank you for your support.
____________________________
Dr. Olaf Selchow
Microscopy & BioImaging Consulting
[hidden email]
[phone]
skype: olaf.selchow

[George McNamara]

*****
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 Olaf, and confocal listserv readers,

Your (Olaf's) survey asks if the respondent (i.e. me) is core staff, but
then ignores the possibility that the respondent (i.e. me) is core
staff. I completed it anyway, though some of my answers involve
'circular reasoning' in that I manage a microscope core and use the
core's microscopes.

FLIM and FCS/FCCS: I managed a Leica SP5/MP/FLIM/FCS&FCCS microscope for
five years in Miami (FLIM from Becker&Hickl, having 2 FLIM PMTs inside
SP5 basically made it less color capable than conventional SP5; FCS&FCCS
from ISS using APDs on X1 port ... in hindsight, I should have marketed
the two APDs some for simple NIRF imaging). The F-techniques were
practically never used, despite my attempts to market them (of course
I'm a biologist, not marketing MBA, and UMiami SOM is ~50th ranked
academic medical center in U.S., not a biophysics powerhouse). My
response may be easy to spot since I added ISS to manufacturers list.
ISS, Leica, etc (I don't try to keep up with the marketing blizzard,
now  have interesting "fastFLIM" stuff on their web sites (ISS with good
explanations and academic publication links, others not so clear).

I encourage you to think about promoting / advising another F-technique:
multiphoton fluorescence anisotropy, especially for drug studies
(BODIPY-drug has high F.A. when bound to its target site [hopefully the
drug company has pretty good specificity in what the drug binds to and
knockout or RNAi can help evaluate this issue; 2-photon F.A. has bigger
dynamic range than 1-photon ... and I seem to recall 3-p even more,
which will become more relevant if/when 1700 nm lasers become more
common, re: Chris Xu, Cornell Univ). See PubMed search hits for:

weissleder r anisotropy

FYI - F-techniques is from title of Liu 2008.
https://www.ncbi.nlm.nih.gov/pubmed/18387308

***

Some suggestions for you in your consulting/advising companies. Note: I
am copying this email to the confocal listserv (I may or not bother to
also post it as a linkedin blog).

This is mostly with respect to the "big four" confocal microscopes -- I
mostly interact with Olympus, Leica, Zeiss, so possible that Nikon has
"nailed the landing" on all (or at least some) of these, I suspect not.

* Confocal microscopes typically cost ~$500,000 (my guesstimate for
median price of point scanning confocal microscopes) but are sold with the:

    ** 2018 equivalent of PC Jr. ... minimal ram, crappy (if any) GPU,
no SSD; typically exactly the same PC sold with new system as whenever
the confocal was introduced. I suggest 'standard of care' confocal
acquisition PC should be something like:

https://www.supermicro.com/products/system/4U/7049/SYS-7049GP-TRT.cfm

          (ok, may not need to be fully loaded with 2 Tb ram, which is
$64K ... but 1 Tb = $32K is reasonable to think about on a $500K
instrument --> ~$560K with this ram plus dual GV100's; no financial
interest in SuperMicro now NVidia [my bank account regrets missing out
on NVidia stock 3+ years ago]).

    ** pathetic computer monitors and graphics cards... hopefully every
confocal microscope at M&M 2018 and Neurosci 2018 will be HD 4K(s) or
better, taking advantage of NVidia Volta GPUs. Sure, two Quadro GV100's
(15 Teraflops single precision, 32 Gb ram each) plus NVLINK2 is $18K,
which is (coincidentally) the approximate price of one TIRF objective lens.

    ** pathetic networking ... I see 100 Gbe (100 Gigabit) Ethernet
cards are available (for ~$800, as are 100 Gbe switches, $10K for 16
ports, $28K for 32 ports, amazon.com list prices). Of course all the
local PC's and file/processing/analysis PCs will need/benefit from this.

    ** pathetic software ... for a couple of examples

           *** Leica's LAS X for SP8 has as crappy image math now as the
TCS software (an Excel macro!!!) had when I managed a SP1 in 2000 (and
the update to "LCS" at end of 2000 was equally bad). Leica and
"MetaMorph" (Universal Imaging Corp --> Molecular Devices) have been
sister companies inside Danaher for many years, still Leica has not
'ported' MetaMorph's Arithmetic to Leica software (nor Stack Arithmetic
either).

           *** Leica's LAS X user interface requires moving the computer
mouse between bottom left ('fast live', only available in HyVolution
mode), mid-left (acquire), top right (channels on/off), mid (contrast).
Sure, Zeiss ZEN Black has about equally crappy user interface --
fortunately I currently have minimal need to deal with it since our
LSM510META is obsolete (and about to be retired!!! - my thanks to NIH
and all the 'confocal' study section S10 grant reviewers for ok'ing our
replacement confocal microscope proposal ... we are going with
instrument written with some 2018 improvements).

    ** Spectral unmixing on Leica SP8 LAS X and Zeiss Black/LSM510:
horribly bad. Most critically: Result channels on either can be in any
order compared to the source images. Applied Spectral Imaging 'nailed te
landing' on this in 1997 and the confocal companies still have not
figured it out 21 years later. I've posted about SUN previously, so for
here I'll just state:

1. get the result channels right.

2. read -- and incorporate:

    Valm et al  https://www.ncbi.nlm.nih.gov/pubmed/27391327

    Hoppe et al  (2008 !!!) https://www.ncbi.nlm.nih.gov/pubmed/18339754 
and https://www.ncbi.nlm.nih.gov/pubmed/27023704  (works on FRET, would
be more useful with more plex).


* New user interfaces: I'm not ready for direct neural USB or 100Gbe
Ethernet, but V.R./A.R., voice, gestures ( not just middle finger up)
would be useful to think about.

* fully loaded image processing, analysis, visualization licenses for
(essentially) free to customer labs and core facilities: the USB dongles
cost $20 each (maybe less). The microscope companies should decide
whether they can really make money on both hardware, bad software, and
support (technical support and field service being different types of
support). I suggest microscope companies should focus on developing and
selling great hardware, 'nail the landing' on acquisition software,
charge reasonable annual fees on annual "service contracts" (software
updates, occasional hardware updates, field service), and
processing/analysis/visualization software that is essentially support
free. Having high quality company software -- with company name in title
bar and logo visible (latter not taking too much GUI space please) would
be a lot better than dumping users onto anti-user software like Fiji
ImageJ and Cellprofiler, and 25 year old MetaMorph (MetaMorph is pretty
good at 2D, atrocious at 3D analysis and the 3D is deficient because
IMA/Measure Objects still has ~64,000 limit of thresholded features ...
to say nothing of MetaMorph not being GPU enabled).

======

With respect to linkedin blogs, I encourage you to check out my blog,
which I wrote because the fluorescence microscopy community (vendors,
core staff, users) are STILL suck at 4 PLEX whereas flow cytometry is
now routinely 12+ plex, the state of the art is 50 plex with 6 way
sorting and Bob Balderas of BD said at a recent seminar will be at 60
plex and 10 way sorting in 3 years (the extra plex powered in part by
adding 320 nm Lasos laser and commercializing Brilliant Deep UltraViolet
['BDUV'] -- and they have full confidence in their R&D process of
achieving these).  Sure, a 5 plex paper was published in 1987,
https://www.ncbi.nlm.nih.gov/pubmed/2444600   with very little follow-up
(except in the tiny niche of spectral karyotyping and M-FISH, see PubMed
spectral karyotyp* ).

I discuss some approaches to 20 plex (plus) fluorescence microscopy at

https://www.linkedin.com/pulse/resolution-blues-meets-21plex-salute-fluorescence-basic-mcnamara/

I mostly think about spectral multiplexing, but note that FLIM provides
opportunities (though suspect data interpretation will be non-trivial).
I'm also intrigued by the potential for CW NIR divide by 2 for Brilliant
Ultraviolets and future BDUVs, given that Stefan Hell published CW 2p
DAPI and Hoechst a long time ago, and Brilliant's absorb a lot better
than single bound DAPI or Hoechst (though an AT-rich DNA molecule with
either bound may have a whole lot of fluorophores in one voxel) ... I
hope to make measurements 'soon').

I sent you (Olaf) a linkedin invitation: if you -- and/or any listserv
readers (whether academic or commercial) -- visit Baltimore for
Microscopy & Microanalysis (Aug 2018), please let me know and think
about visiting our image core for (part or all of) a day, before or
after the meeting.

More thoughts -- and lots of awesome tables -- about light microscopy in
our Unit (open access, so no direct financial interest; I do encourage
you to read online or at least print out double sided to save some
trees, and be sure to also check out the supplemental appendix)

http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract

No need to print out figure 1 for your desk, you can download the image
directly from http://home.earthlink.net/~tiki_goddess/TikiGoddess.jpg 
(looks great on HD 4K).

enjoy,

George

p.s. your original post was clear in intent - I do not think you
need(ed) to apologize to anyone.


On 4/12/2018 3:02 AM, Olaf Selchow 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.
> *****
>
> *** Please note that this is a commercial posting - or maybe a kind of "semi-commercial" posting ;-) ***
>
> Dear Ralf, dear fellow listers,
>
> Please apologize that I didn't mark my earlier posting appropriately. This was a mistake that should not have happened.
>
> Still, please let me explain that I would like to ask you all for the favor to support my information gathering with your feedback in the below survey on Advanced Microscopy Techniques (FLIM, FCS, SMD etc).
>
> Yes, I am making my living as a professional microscopist ... and will use the information for my professional activities - as probably most of you do in one or the other way. And yes, I agree that I should have made this more transparent. On the other hand, be assured that I am not intending to sell you anything. If I am doing my job right, I am advocating your interests and requirements with commercial manufacturers and suppliers of microscopy equipment and software.
>
> I have been asked by some of you about "what would be in for you" with this survey. Well, I am a one-man show and I don't have the ressources to incentivize participants with anything else but the promise that I will use the feedback to advocate your requirements with those companies that I have the chance to work with.
>
> Please, again, accept my apologies for not making the nature of my posting very clear in the beginning.
> With best regards,
> Olaf
>
>
> ========
> Please find the survey following the link          https://goo.gl/forms/b7J32fAgqvvBaw452
> ========
>
> Thank you for your support.
> ____________________________
> Dr. Olaf Selchow
> Microscopy & BioImaging Consulting
> [hidden email]
> [phone]
> skype: olaf.selchow

--


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