Posted by
Tim Feinstein on
URL: http://confocal-microscopy-list.275.s1.nabble.com/shopping-live-sample-confocal-super-res-tp7590816p7590823.html
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I'd follow on George's comments about Leica with a couple observations. STED does seem excellent for fixed cell imaging, but the iterations that I've used had to make trade-offs with live cell imaging due to speed and the potential for photodamage. Stefan Hell has published some improvements in live cell STED, so I'd be interested to see how that has translated to commercial systems.
Note that Leica's implementation of spectral detection doesn't work in real time like Nikon or Zeiss, which use 32-PMT arrays, so it's hard to get as creative with live cell multiplexing. On the other hand Leica's implementation of FLIM in the FALCON and Stellaris is interesting and could supersede the limitations of spectral detection. Lifetime imaging *REALLY* expands the system's potential applications. Ammasi Periasamy at UVA has been promoting FLIM for a long time but it's been a pretty arcane technology, and I'm curious to see how it gets adopted now that there's an accessible consumer model. I don't have any commercial interest, just a general interest in the technology. A demo that I attended where we did live cell acceptor photobleach FRET looked promising.
All the best,
T
Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of George McNamara
Sent: Sunday, April 26, 2020 3:45 PM
To:
[hidden email]
Subject: Re: shopping: live-sample confocal+super-res
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Hi Jeff,
I suggest you extend the list to Leica Stellaris confocal STED, full load of their new POWER HyDs internal, plus as many more POWER HyDs (some NIR, i.e. GaAs) on X1 port. White light laser (WLL: modest average power, but pulsed so much higher ... can also add nearby ex wavelength, ex. 504 nm + 496 nm + 488 nm for AausFP1). Hopefully also 405 nm laser for BV421 and all the BVs (and SuperBrights ... and QDots, if ThermoFisher has solved QD-antibody aggregation issues). Probably a reach, but Leica (since they've been pushing WLL for many years) and maybe the other microscope companies, should be looking to get back into the UV with WLL + SuperK EXTEND-UV
https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nktphotonics.com%2Flasers-fibers%2Fproduct%2Fsuperk-extend-uv-supercontinuum-extension-unit%2F&data=02%7C01%7Ctnf8%40PITT.EDU%7C0f01b4ce8955456b667608d7ea1b431a%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637235275124621732&sdata=cU3aOnJAH92y9NXNur7hAGsm1hue%2B%2BAUXroujUpOcl0%3D&reserved=0especially for BUV395 and the other BUVs (and I suppose QDots).
***
A lot of live cell experiments are going to involve fluorescent proteins, so:
AausFP1 green fluorescent protein should finally obsolete EGFP ... 5x brighter than EGFP, ~2x brighter than mNeonGreen ... and much narrower excitation and emission spectra (so ideal for buying WLL) ... and for many uses, could be tandem dimer, so double those. Amino acid sequence in figure in text, DNA sequences (not human codon optimized) in supplement
https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.biorxiv.org%2Fcontent%2F10.1101%2F677344v2&data=02%7C01%7Ctnf8%40PITT.EDU%7C0f01b4ce8955456b667608d7ea1b431a%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637235275124621732&sdata=EbkR%2FLXS9IqZJsLg%2FdRLt%2F8bLophTr%2BhcGwx68qHlQY%3D&reserved=0Nathan spoke at JHU May 2019: has yellow version (so even more value in
WLL) ... also has new generation other colors (but I've not seen further bioRxiv preprints or publications).
enjoy,
George
p.s. if not your core, some core at NIH could buy an Abberior Instruments MinFlux ... 2 nm (and 2 color) "real time" precision localization,
https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.abberior-instruments.com%2Fproducts%2Fminflux%2F&data=02%7C01%7Ctnf8%40PITT.EDU%7C0f01b4ce8955456b667608d7ea1b431a%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637235275124621732&sdata=MFsir5oLR4VwEzxdnbs912RBa4w%2BwYif3fDNqqsrTWs%3D&reserved=0hopefully will come down in price in next couple of years, but the biology in the meantime could be worth it.
On 4/26/2020 3:06 PM, Reece, Jeff (NIH/NIDDK) [E] wrote:
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>
> Dear List,
>
> We are a core facility ready to make a major purchase, seeking advice. The system needs to provide fast, live-sample confocal imaging, but also super-res in the 100-150nm range (xy). Here is a sampling of the applications we are trying to satisfy:
>
> 1. Z-stacks of cultured cells over time, multi-color labeled. Super-res and standard confocal.
> 2. Z-stacks and/or time series of live tissue/organisms (e.g. c. elegans, oocytes) up to 40 microns deep (at least), multi-color labeled, super-res and standard confocal.
> 3. Z-stack, tile and stitch, super-res of fixed samples, e.g. FISH and tissue slices (e.g. mouse kidney).
>
>
>
> We narrowed it down to the following instruments:
>
> 1. Nikon W1 SoRa spinning disk
> 2. Olympus W1 SoRa spinning disk ("SpinSR")
> 3. Visitech vt-iSIM (VisiView software seems to be the best choice here in the USA?)
> 4. Zeiss LSM 980 AiryScan 2
> 5. Zeiss Elyra 7 Lattice SIM
>
>
> I will send another email for those that are theoretical-minded; for this email, I am interested in practical, hands-on impressions.
> For any of you that have compared any of the above systems, I would greatly appreciate to hear those impressions, either to the list or directly to me.
> Here are some common categories of comparison that may jog your memory and/or provide a framework for your response:
>
> 1. Resolution;
> 2. Speed;
> 3. Sensitivity;
> 4. Photobleaching;
> 5. Maintaining focal plane over time (all the vendors do this well now?);
> 6. Color-correction from blue to far red, to edge of image field;
> 7. Usability of software - i.e. user-friendliness, appropriate for a core facility;
> 8. Functionality-- i.e. range of features; capability to do what you need from a workflow/experimental point of view;
> 9. Reliability, robustness of the system;
> 10. Customer support level.
>
> Stay Safe and Healthy,
> Jeff
>
> Jeff Reece
> Ph: +1.301.451.4330
> E:
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