Re: shopping: live-sample confocal+super-res

Posted by Alison J. North on
URL: http://confocal-microscopy-list.275.s1.nabble.com/shopping-live-sample-confocal-super-res-tp7590816p7590844.html

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Hi all,
Interesting discussion.  I just want to address a few specific comments regarding the vt-iSIM system that have come up in other people's posts today.  I spent an entire year trying to decide on the configuration of my iSIM, so hopefully some of this will be helpful info.

1) Sadly Gabor, you cannot run the iSIM through Elements if you are in the USA.  Nikon does not support it over here. Don't get me started on this...

2) Pavak is absolutely correct that if you are imaging thick organisms like zebrafish or worms, the Silicone objectives will be your best choice, and that means either Olympus or Nikon.  HOWEVER, Silicone objectives only work well if you have adjusted the correction collar absolutely perfectly - and this is something that is difficult to do manually without experience, or even (on some systems) if you have lots of experience, due to the objective being hard to access on an inverted system with lots of "stuff". I should know, I have often spent up to 2 hours "nudging" the collar of the Si objective on my OMX system until it is OK (due to the lack of eyepieces), and that is a painful process.  I have spent years urging Olympus and Nikon to motorize the correction collars on their Si objectives, but no luck with it yet, even though Nikon HAS done so for the 100x oil objective.  (Please help me push them on this!) Therefore in the end I chose a Leica stand for my iSIM, using the 93x glycerol objective that DOES have a motorized collar.  Although glycerol is not quite as good an RI match as Silicone oil, we feel that a well-adjusted collar on a glycerol objective works better than a sub-optimally adjusted Si objective, and therefore this is perhaps a better choice for a core facility.   Not that this helps if you mainly work at 60x, as Pavak does, since it's only the 93x objective that is motorized.

3) Regarding the need for a separate FRAP/ablation device at additional cost, I am actually pretty excited with my iSIM system set-up - we have a triple fiber output so that a single set of lasers can be used for the iSIM, the Mizar TILT light sheet module, and also a VisiFRAP module.  These are all controlled by the VisiView software. It has taken quite some months to get everything working properly at full speed and triggering of two cameras etc. etc. , and I haven't yet tested the VisiFRAP module as that was about to be set up when they closed down the University, but the software is certainly allowing super-fast dual colour iSIM and Mizar imaging.  I cannot WAIT to get back to the lab and start using it to image some virus...

All the best,
Alison

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From: Confocal Microscopy List <[hidden email]> on behalf of Cammer, Michael <[hidden email]>
Sent: Tuesday, April 28, 2020 3:16 PM
To: [hidden email] <[hidden email]>
Subject: Re: shopping: live-sample confocal+super-res

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Has anyone mentioned yet that if anyone needs to do localized photoactivation, bleaching, ablation, with spinning disk or ISIM you need an additional unit like the Bruker miniscanner with additional lasers and matching filter blocks etc?


Laser scanning confocal may be better for this.


But the white light laser for Leica, which otherwise is an incredible confocal, may not be strong enough?


For most live work, ISIM or spinning disk way better.  Exception would be for yeast and bacteria where we like to zoom in and over sample.


Cheers-


Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory

NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016

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From: Confocal Microscopy List <[hidden email]> on behalf of James Kerin <[hidden email]>
Sent: Tuesday, April 28, 2020 10:39 AM
To: [hidden email]
Subject: Re: shopping: live-sample confocal+super-res

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Dear Jeff,

If you are considering spinning disk configurations with super-resolution capability then another combination to consider is the Crest X-light v3 spinning disk confocal together with the Gataca Live-SR. The v3 of the X-light is a great and cost effective low light confocal imager, couple this with the equally cost-effective Live-SR for the optical modulation to achieve super-resolution imaging. The combo will take you down to around 120nm resolution.
This recommendation is made from an honest belief that you should take a look and give serious consideration, but please note I have to declare a commercial interest as Cairn use the Crest X-light and Gataca LiveSR in our configurations in the UK and Europe.

https://urldefense.proofpoint.com/v2/url?u=https-3A__crestopt.com_xlightv3_&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=VGygx11l5BTnh0tin0WYmhlIVJwDAlMSr74ybuxIzUE&s=c3vRjzCudBMPGVJI2zA3jz6uv94ANofm-F10pXNVEOs&e=

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.gataca-2Dsystems.com_&d=DwIFAw&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=VGygx11l5BTnh0tin0WYmhlIVJwDAlMSr74ybuxIzUE&s=kaf25E_h6snd0qfol8M6DgwhvSCiE2G3HtBDQ_zkvK0&e=

Martyn (on James' email account)


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On 04/26/2020, 08:06pm, "Reece, Jeff (NIH/NIDDK) [E]" ([hidden email]) 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

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