NetNotes
I tested the Andor iXon-897 EMCCD and the Prime 95B, both
specifically for single-particle tracking (SPT). I think they both performed rather well, and I honestly don’t think you can go wrong with either (others may feel differently), but your conditions may make you lean one way or another. If field of view is important to you, I would recommend the Prime 95B. If, however, like me you need some extra sensitivity when you have weak fluors (smFRET in my case), then the Andor EMCCD might be your best bet. Krishna Mudumbi
krishna.mudumbi@
yale.edu
As a 95b user I confirm that sCMOS is an excellent choice.
However, if you use a sCMOS for single molecule localization, then I suggest you keep in mind that traditional single-molecule protocols assume a uniformity of noise that you find in CCD and EMCCD chips but not sCMOS chips. Make sure you consult the vendor and check the literature on this, for example, Mandracchia et al. (2020), Nature Comms 11: #94, described how to minimize artifacts with sCMOS. I believe the magnitude of the non-uniformity is less of a concern for most users, but single-molecule tracking demands a lot from a chip. Timothy Feinstein
tnf8@pitt.edu
We were drawn to the sCMOS mainly because of the large (very
large compared to an 897) field of view. Yes, the sCMOS isn’t as sensitive as EMCCD in the super low photon/pixel limit (at least on paper), which is why I’ve reached out to get some opinions. We currently don’t use any binning on the EMCCD. I should mention our CEMCDs are ∼10 years old so I’m sure they aren’t as sensitive as they used to be. It sounds like we would be okay replacing an EMCCD with the sCMOS, but I’d still like to hear from someone who has done single fluorophore tracking with a sCMOS and how performance compared to EMCCD. Jeff Spector
jospector@gmail.com
I ordered an Andor iXon897 just a few days back. I asked the same
question, researched, talked with many experts, talked with vendors from both Hamamatsu and Andor, and tried to understand the latest technology. Finally, I decided to buy an EMCCD. BTW, I have used several EMCCDs extensively in the last eight years, specifically for single-molecule tracking, single-molecule FRET, and ion-sensing at diffusing conditions. Here are some of my personal opinions/thoughts from the perspective of an extensive user of EMCCD. (1) EMCCD is still the gold standard for single-molecule tracking and single- molecule FRET experiments. A high frame rate with high sensitivity is essential for a diffusing single-molecule to get a complete track- patch (dynamics) with a small step size (time/frame). Here, sensitivity is critical and sCMOS does not match EMCCD for single-molecule tracking with fast dynamics. You can go to a high frame rate only if the camera sensitivity is increased. You may get tracking data with sCMOS, but I would assume it would be at a much lower frame rate to reproduce similar quality data. Or, you have to use a high-power laser to get a fast frame rate, which can cause rapid photo-bleaching. (2) If protein/ DNA conformation dynamics are relatively slow with immobilized conditions, then sCMOS and EMCCD will produce similar results because recording of data is with a slow frame rate. (3) Tere are recent publications that study single-molecule FRET using sCMOS, where the proteins are fixed on glass or cell/virus membranes and dynamics are slow. Tey take advantage of a large sensor, which can generate tons of data/time-trajectories in a single FRET experiment. Many groups even use automated algorithms to generate time trajectories of donors and acceptors. When a molecule is fixed and the dynamics are slow, sCMOS is fine. (4) Ca2+
ion-sensing data is very hard to get with a sCMOS, at
least in our case with T-cells. (5) QE is the same (∼95%) in both cases. I would consider critical applications to decide EMCCD vs sCMOS; fast vs slow dynamics or conformational changes. Overall, EMCCD is still
2021 May •
www.microscopy-today.com
the best for single-molecule experiments including diffusing single- molecules, in my opinion. Dibyendu Sasmal
sasmal@iitj.ac.in
I thought I’d wait for the users of the various cameras to comment
on their experiences. From our side, as manufacturers of the iXon 897 models, we can make some comments from what we see. As others have mentioned, it comes down to imaging priorities and optical matching. Te iXon 897, although now an older EMCCD model, should still perform very well. Tat model did have our RealGain and EMCal features that helped avoid issues with EM gain aging. I doubt there would be any issues from EM aging affecting performance, and if it still cools as well as it did, then we see these models continue to give comparable performance to when new. Te newer Ultra 897 EMCCD models, of course, have been updated and have revised electronics for lower internal noise, which helps with very low signals and most notably the increased speeds. Te EMCCD cameras still give the best sensitivity, so when you need that there is no equal. To maintain similar sensitivity, but improve the field of view, an option is the Ultra 888 model (or other EMCCD models that use the larger 1024x1024 sensor), that gives a much larger sensor area the same size as the 4.2 megapixel sCMOS format, albeit using a 13 μm pixel size. We find many customers with the older iXon models oſten go that route over back- illuminated sCMOS when demoing for single molecule experiments due to the hit in sensitivity they would take. For the back-illuminated sCMOS, the main sensor used is the GS400BSI and our camera model with this sensor is the Sona 4.2B-11. If you have enough photons you can potentially get up to 32 mm field of view (and higher speeds). So, it can offer a big benefit for some. Testing is always recommended as there are too many subtleties in parameters from labels to optics involved. Alan Mullan
a.mullan@
andor.com
Chemical Fixation Before Plunge Freezing 3D EM Listserver What chemical fixation protocols work for your projects before
plunge-freezing (or high-pressure freezing (HPF)), or what types of chemical fixation were not good (destroyed more than it helped) prior to cryo-EM sample preparation? I am interested in your experiences and if you have addressed this question in an article and would like to share the link with me. For cryo-EM we prefer to work as close to native as possible, avoiding chemical fixation, but sometimes fixation is needed to test or move on with tricky projects. For example, when bacterial cultures are tricky and samples need to be stored, or when biosafety restricts work in the EM labs (lab specific and different). Linda Sandblad
linda.sandblad@
umu.se
I have used 1% glutaraldehyde (GA) for fixing BSL3 viruses
for cryo-EM. Resultant single particle resolutions ranged between 7–9 angstrom. A have also used 2% GA while fixing BSL3 virus- infected cells for HPF followed by thin section TEM. If you are fixing mammalian cells intended for HPF, I would very strongly recommend not centrifuging them for >500 g at any time during fixation or further washes, even ∼200 g if you can go that low. GA helps to preserve ultrastructure, but I would recommend leaving the sample in the fixative only for the minimum essential amount of time. Tere are several reports out there with time recommendations. If you need further details of the protocols here are links:
1)
https://urldefense.com/v3/__
https://www.frontiersin.org/arti- cles/10.3389/fcimb.2020.580339/full__;!!Mih3wA!TMDW6o_ cpZIwgUlq8Ekweus0f tgX0bj6FrhPvLkrP1M30CrZe6d64_ bZErBdSi4cVA$ - for HPF.
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