NetNotes


2) https://urldefense.com/v3/__https://www.sciencedirect.com/ science/article/abs/pii/S0166093419304173__;!!Mih3wA!TMD W6o_cpZIwgUlq8Ekweus0ſtgX0bj6FrhPvLkrP1M30CrZe6d64_ bZErBQcnm7Kw$ - for inactivation of viruses intended for cryo- EM/cryo-ET. I have never used paraformaldehyde and I have never done CLEM, so I don’t know how that would affect either fixation or fluorescence experiments. Amar Parvate aparvate@lji.org


Cryostorage Concerns 3D Listserver We are seeking a good solution for ice-free storage of vitrified


samples. We use a Vitrobot and front-loading TEMs only, so no autogrids. Currently we place the grids in a plastic sample puck and then into a 50 mL Falcon tube with storage in a Worthington 35LDB LN2


dewar. We see


some frosting of the polystyrene lid and dry this periodically to minimize frost. Our lab is controlled to 40% humidity, which unfortunately we cannot change. Are there any good off-the-shelf solutions for ice-free storage that we are missing? John Watt watt@lanl.gov


You won’t find a solution for ice forming on the polystyrene lid. I


think the majority of us have this issue. But do you see ice contamination (small ice flakes) in the 50 ml Falcon or next to the grids in the sample puck? Te bottom of my dewar has lots of ice flakes, but I do not see ice flakes in the sample puck, so I do not really care about it. I also use 50 ml Falcon tubes and I punch holes through the lid. I almost never get ice flakes in the Falcon tubes. Te humidity is not controlled in the room, so it varies a lot. Sylvain Trepout sylvain.trepout@curie.fr


We have found that the primary time grids are susceptible to


ice contamination is during the vitrification workflow itself, and not during storage. Generally, aſter grids are vitrified, placed in a grid box, and the grid box closed with a cover, they are well-protected from contamination. As grids are plunged and then transferred to the grid box, they are sitting in the larger LN2


dewar. Te styrofoam ring (used to collect a pillow of cold, dry gas for the grid transfer from liquid ethane to LN2


of condensed ice, as it is cooled from sitting atop the LN2


reservoir of the Vitrobot styrofoam ) oſten collects large amounts


new grid is plunged, and the dewar moves up flush against the bottom of the Vitrobot box, that ring is submerged fully in the LN2 crystalline ice gets released into the LN2


. Every time a and condensed


where the prepared grids are


sitting. We have found this to be the major source of ice contamination. Michael Godfrin mgodfrin@nanosoſtmaterials.com


Spinning Disk Comparison Confocal Listserver Has anyone compared the emitted light gathering capabilities of a


Yokogawa CSU-X1 vs a CrestOptics X-Light V3? Neither have a micro- lens array on the emission path, and both units would have 50 μm diameter pinholes with 250 μm spacing. Techs from a large microscopy company said the CSU-X1 is superior for fast live-cell imaging in cell culture, but it’s not clear to me why. Is Yokogawa able to get a higher density of pinholes on their disk? Am I misunderstanding how pinhole spacing works? Tanks in advance, William Giang wgiang@emory.edu


Te CSUX1, being a dual-disc spinning system, has micro lenses on


the excitation side that enhance the excitation efficiency. Te CSUX1 is laser-based and has the ability to synchronize disc rotation with respect to exposure time of the camera (1800 rpm or 5000 rpm). Whereas Crest offers a simple single Nipkow Disc without micro lenses, and more importantly it uses bright LED light, if I am not wrong. Naturally the CSUX1 should perform better. Ganesh Kadosoor ganeshkadasoor@gmail.com


80 Te rationale is simple. Te purpose of the spinning disk is to


attenuate out-of-focus light. If the CSU-X1 attenuates this unwanted emission by a factor of 30 (estimate, I didn’t do the math), it will also attenuate the laser excitation by the same factor. With a strong laser that shouldn’t be a problem, right? Well, 1:30 pinhole crosstalk may not be enough. If you bleach a (small, let’s say 1 μm by 1 μm) cone of light in a thick fluorescent layer you won’t be able to see it with the X1, but you can see it clearly with a point scanner. Tat’s why the CSU-W1 and Dragonfly have sparser pinholes and higher attenuation. Without the microlenses you would end up with very little excitation, even with powerful lasers. And as a matter of fact, we use quite strong excitation (30% with a 150 mW laser) to capture a z-stack quickly and do longer pauses between z-stacks. Tis helps with motion artifacts and allows for deconvolution. Back to the original question. With identical pinhole pattern and overall optical configuration (minus the excitation pinholes), the detection efficiency should be the same. Te ultimate limiting factor will be how much light can the disk handle, how you deal with the reflected laser light from the disk (essentially 100%), and the autofluorescence of any element that is common to the strong excitation (before the disk) and emission paths. Zdenek Svindrych zdedenn@gmail.com


As Zdenek says, quite substantial laser powers are needed


under some scenarios without the microlenses concentrating the energy. The cost for high-power versions of certain lasers can also be quite high, if the required energy density is available at all, so on occasion a weaker laser is the only option. A lower-power laser also requires less heat sinking and electrical current, which simplifies the overall design and, in some cases, can lead to longer laser life. Craig Brideau craig.brideau@gmail.com


As some of the responses so far have indicated, there are variety of variables in design that will impact performance. Given the forum


www.microscopy-today.com • 2021 May I have never understood the rationale for micro-lenses in a


spinning disk system. Tese systems are aimed at live cell imaging. I have never used anywhere near full laser power on either our Yokogawa system or our Crest system. Cells do not tolerate high excitation light in general, and so we always work with the minimum light level possible and that is never near the limit of laser power. So, I don’t see the rationale for the added cost of a micro-lens system, unless you have a really low-power laser source. I would be curious to know if anyone has a different opinion. As to the question that was asked, I have not directly compared the two, but not sure how I would since they are on different microscopes, objectives, cameras, laser launches, etc. I can say that both give nice images of live cells. I like the ability of the Crest to function in confocal or non-confocal mode, which our Yokogawa system cannot do. Dave Knecht david.knecht@uconn.edu


I’d like to point out a few key points on the CrestOptics solutions,


especially the X-light V3. 1) Yes, there are no micro-lenses to focus excitation light, however, this is compensated by the use of more powerful laser sources (cost-effective multi-mode sources) to achieve comparable excitation power on the sample. 2) On the X-light V3, micro-lenses are employed to achieve homogeneous illumination, so that quantitative imaging can be done on the full field of view (FOV). Tis FOV measures 25 mm, meaning much faster data collection (more cells in a single FOV). 3) Two cameras with FOV 25mm can be used simultaneously for even faster data collection. 4) Te CrestOptics disk spins at 15k RPM, meaning acquisition speed >1kHz without artefacts. 5) Te disk pattern can be customized for the best ratio of confocality and throughput depending on the sample. Alessandra Scarpellini scarpellini@crestoptics.com


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