NetNotes

As most vendors are now providing more laser lines past 640nm, I was wondering if anyone had found or developed slides/tools for performing QC checks in the 700–800nm ranges? I'm thinking of field illumination, point spread function (PSF) beads and image quality tests. I would be really interested to hear what everyone is doing in this space. Thanks. Paul McMillan [email protected] ArgoLight spectra provides emission data out to 800nm (https://argolight.com/power-hm-v2/). The absorption spectrum is low at 700nm and lower at 750nm, but above zero (compare to trace at 800nm and at 200nm). You can probably get more data from the company. Plan B (for beads): several vendors, including ThermoFisher, Spherotech, and probably others sell various fluorescent microspheres, mostly for the flow cytometry market. You might look up their near infrared fluorophore products. For experiments in aqueous solutions, switching from H2O to D2O (about $0.10 per 100µL from MilliporeSigma) increases fluorescence of many red to NIR fluorophores by 2× to (around) 5×, see Cy5.5 Alexa Fluor 700, Cy7, DY749P1 (Figure 2G in J Maillard et al., Chem Sci 19 (2020) https://doi.org/10.1039/d0sc05431c). George McNamara [email protected] OptiSlides will be launching NIR slides in February. We’ve partnered with Biotium and are using their NucSpot® 750/780 nuclear dye and their CF® 790 and CF® 800 secondary antibodies. These NIR channels are in addition to the 4 traditional colors. An example can be found here: https://imgur.com/a/HDmrdRZ. If you need something now, our Stone Pine slide autofluoresces from the UV to the far red and into the near infrared, though the signal starts to wane the further into the NIR you go. OptiSlides are available in Canada and the US directly through Luxidea (http://optislides.com) or via Cedarlane labs. Slides are also available at Cosmo Bio in Japan and will soon be available in the UK and EU through VHbio. We don’t have a distributor in Australia and New Zealand yet, but drop me an email and we will see what we can do! Kamala Patel [email protected] You do not specify your microscope system, but if it allows for reflection (for example, reflection confocal) you should be able to use gold beads for PSF measurements, as in the visible spectrum. For field uniformity, a mirror might do the trick. Steffen Dietzel [email protected] I would like to colorize erythrocytes in SEM images for a publication. I have no experience in colorizing B/W images. Is there an easy way to do it with free tools? Stephane Nizet [email protected] I would create a mask for the erythrocytes in Photoshop or other image editing software. With a mask created you can add whatever color you want for the erythrocytes and another color for the background. The precision of the mask will directly relate to the overall quality of the final image. And you may want to slightly blur the mask edge, so the transition fits the overall sharpness of the image. Oh, and it WILL take some time to do it right! David Scharf [email protected] Old, old school. Back before digital, we printed out the black and white micrograph from negatives and used highlighters or markers to color. You can then take a digital picture. Not so fancy, but it works and is fast. Paula Keene Pierce [email protected] The way a lot of people give false color is to use different detectors and assign each image of the same field of view to a different color channel. Nathan McCorkle [email protected] We used to do this with Photoshop. Sometimes we'd use different filters (like an FFT lowpass in ImageJ) combined with masks for different color channels and the outline. There's no one right way to do it, but it involves artistic skill with whatever tools you decide to use. You won't know until you play. Curious what would happen if you loaded the grayscale image into one of the new AI image generators and directed it to keep the high contrast edges intact and to colorize it. If the AI were trained with grayscale pictures which were then colorized, it might work or get you part of the way there. Michael Cammer [email protected] Microscopy Australia has a YouTube video showing how to colorize an SEM micrograph using Photoshop: https://www.youtube.com/watch?v=7vA8sAvPIaA&t=32s. PhotoPea is a great free alternative to Photoshop: https://www.photopea.com/ Susan Warner [email protected] Instead of Photoshop, structures can be segmented far more accurately using an AI tool like Dragonfly. I’m not sure if this is more efficient as I did not compare the two. Dimitri Scholz [email protected] In the end, Photopea worked well for colorization of SEM images, very easy to use and it did not take too much time. I used the magnetic wand to mark the edges and just added some color. Thank you, Susan, for the advice! One word of warning on the use of AI for colorization: I asked co-pilot to identify erythrocytes present in an SEM image and to colorize them in light red. At first sight, the result was tremendous with very impressive images. But on closer observation, I noticed that the AI added perfect-looking erythrocytes to my image, which were not present on the original image! Moreover, the AI colorized some additional structures, and I cannot make it remove the color from them. Also, with each iteration, the AI made the red color stronger, even though I forbade it to do it! So, let's be careful with AI. We are keen on using AI to sell products, but they may be less intelligent than some want to make us believe. Stephane Nizet [email protected] We have lost a 50ml Falcon tube in our cryo dewar. Is anyone aware of a good claw that can retrieve it? Thanks. Reza Khayat [email protected] Your best bet is probably to empty the entire dewar. Large styrofoam boxes are very good for this sort of thing. David Morgan [email protected] Alternatively, you can buy very long crucible tongs (∼ 20 inches or longer) and hope to get lucky. If you empty out all the cylinders from the dewar and wait for a few minutes you should be able to get a good view with a flashlight to find its location and get it out. Axel Brilot [email protected] We have used the: Pick-Up Tool, 33-inch length by Trash Gator (https://www.amleo.com/trash-gator-pick-up-tool-33in/p/GT33) to fish out a puck that was dropped in a dewar. Bill Rice [email protected] We managed to retrieve a 50ml Falcon tube (with great difficulty) using something similar (you can get it on Amazon). Anindito Sen [email protected] Emptying the dewar into a Styrofoam-type container works well, as David Morgan suggested. When the liquid level is low, I swirl the dewar and then quickly turn it upside down into the foam container. I have been able to retrieve all lost tubes and boxes this way. David Belnap [email protected] We are troubleshooting an issue where our glow discharger(s) that seem to be of variable efficiency (sometimes making carbon grids ­appropriately hydrophilic, sometimes less so, occasionally not at all). My current theory is that the ambient humidity of the room is affecting the efficiency of the glow discharge. Has anyone encountered this or looked into it more thoroughly, and if so, what was your fix (Account for humidity in timing/current? Move the glow discharger into a more humidity-controlled room? Desiccate the air going back into the chamber…somehow)? Other variables that have been ruled out are a faulty glow discharger, location of the grid in the metal block, and whether it is the first glow of the day session. We regularly have >50% humidity in the room during the summer, even with the dehumidifier running at full-speed, because of air flow, etc., variables in the room that we cannot change. We have two glow dischargers, a Pelco easiGlow and a Quorum/EMS GloQube, and have seen this with both dischargers, although we use the GloQube more, so we have seen it more with that. At one point I tried to blow all the carbon off a grid with a very prolonged (5min @ 20mA) glow discharge on the GloQube, and all the carbon was still entirely intact, which made me a bit nervous. Relatedly, how often and how do you clean the metal blocks that grids glow discharge on? We were thinking of using acetone and/or alcohol but would appreciate recommendations! Talya Levitz [email protected] Recently (∼1-hour) discharged grids have always worked for me, if I observe the glow did take place. However, at times I suspect that ambient humidity is affecting how long my grids remain hydrophilic. Could a time component explain what you're seeing? Personally, I only clean my grid blocks if there is something on them (for example, plastic residue after chloroform washing), or if I am sterilizing them because the grids will be used in cell culture. Based on my observations the mode for other users is “never” and many people glow discharge on the same parafilm-wrapped glass slide for years and years. Ethanol, isopropanol, acetone, or chloroform all seem like reasonable solvents to me, depending what might be on the block. I also doubt if a long 20mA glow is expected to destroy a quantifoil film. I usually do 15mA for 45 seconds and observe no significant damage even if the grids are processed multiple times. But I haven't tried to go all out. Daniel Asarnow [email protected] Thanks, Daniel. In our case we can see that hydrophilicity is an issue immediately, although it will decrease over time. We start to see in grid quality after minutes of out in the air which is than I have seen in other labs. I if whatever is the decrease in overall hydrophilicity is also to the length of time the glow discharge can to clean our and We do with it glow discharge but people it with their so I'm sure there is on from that. to know the film. easiGlow we occasionally use the at much lower time than minutes at but it to destroy carbon because it is very Talya Levitz [email protected] to this from a of need chloroform We have also had users not the all the way and then it glow an you can hear the can on the Pelco if the is out. 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