NetNotes


As a practical matter, this issue is resolved by the patch


approach. How cryoSPARC averages the patch fit quality has not been described, but tilted images can be filtered very well in my experience. Te local defocus patch estimates are also generally very accurate - it’s common for CTFFIND to fit a highly tilted image to 6-8°


, but patch fits of the same image extend to 3-4° (the


programs use the same definition of the fit resolution), and single- particle estimates improve on the patch values for large particles like ribosomes or the 20S proteasome. Tese notes are all from a single- particle perspective. Daniel Asarnow asarnow@msg.ucsf.edu


Tis paper from 2007 in Ultramicroscopy, “Te contrast-


imaging function for tilted specimens” (https://doi.org/10.1016/ j.ultramic.2006.07.010) seems relevant to your question! Michael Elbaum michael.elbaum@weizmann.ac.il


Tat’s the paper by Ansgar Philippsen et al. I mentioned. Te


theory presented there is only valid for thin specimens, but it is not limited to small patches, so it describes contrast “transfer” (not a transfer function!) for the whole image. Philip Koeck koeck@kth.se


Tanks to all for their input. Prodded by several of you, I


realized that I was trying to remember a program called ctſtilt that was released in parallel with ctffind3 many years ago. From the responses I received, it appears that goCTF and the version of ctffind in the development branch of cisTEM are more recent versions that do what ctſtilt did. It is also clear that some packages have built- in ways to process single particle data acquired from a tilted grid. I was looking for something to evaluate the images themselves, before doing any further processing. I did not try any of these packages, and it is entirely possible that they generate diagnostic data similar to the results from ctſtilt (and the others listed above). Again, thanks for all your input, and I hope this summary isn’t a waste of your time. Daniel Asarnow asarnow@msg.ucsf.edu


New Spectra Viewer Confocal Listserver Dear all, I would like to draw your attention to our new fluoro-


phore database and spectra viewer. You might be wondering why we need yet another spectra viewer, but I think we included some very useful features: * Te viewer uses a large database with more than 550 fluo-


rophores and spectra of more than 250 optical elements (filters and mirrors). * Te database contains two- and three-photon excitation spec-


tra for more than 150 and 10 fluorophores, respectively. We are regu- larly adding additional spectra. Please let us know if you have spectra that are currently not included. * Custom spectra can be uploaded and displayed along with the


spectra contained in the database. * Plots can be displayed using normalized or absolute values on


a linear or logarithmic axis. * In addition to the “standard” spectral plots the app provides a


“2D viewer” in which excitation and emission spectra are combined into 2D contour plots. Tis is particularly useful when displaying excitation/emission spectra for various fluorophores simultaneously. * Several additional fluorophore properties are contained in the


database and can be accessed through a fluorophore browser (a table with various search and filter functions) or visualized using a 2D scatter plot. Up to four properties can be visualized simultaneously using the marker’s x- and y-coordinates as well as their size and color.


2022 May • www.microscopy-today.com Tank you for sharing. It looks good. One suggestion: could


you link with the fluorescent protein (FP) database (https://www. fpbase.org)? Tat resource has information about each FP, includ- ing a unique identifier. As part of QUAREP.org we are working to revise the Microscopy Metadata specifications developed initially by 4DN-BINA and OME which were recently published on Nature Methods (https://doi.org/10.1038/s41592-021-01327-9). Caterina Strambio caterina.strambio@umassmed.edu


Tis is awesome and very helpful. I am wondering if we can


also start including other molecular (for example, opsin’s) spectra in such databases. Tat will be a great resource for optogenetic experiment/setup designing. Anuj Sharma anujjnu@gmail.com


Along the same vein of discussion, I thought this would be a


good place to post links to the many different spectra viewer tools online that I’ve found:


1. Termo Fisher Scientific SpectraViewer: https://www.thermofish- er.com/order/fluorescence-spectraviewer#!/


2. Omega Filter’s Curvomatic: https://www.omegafilters.com/curvo- matic


3. Max Planck Institute Spectra Viewer: https://public.brain.mpg.de/ shiny/apps/SpectraViewer/;


4. Chroma’s Spectra Viewer: https://www.chroma.com/spectra-viewer 5. FP Base (fluorescent proteins only): https://www.fpbase.org/ spectra/


6. Semrock’s Searchlight: https://searchlight.semrock.com/ 7. Fluorophores.org (click on dye to see spectra): http://www.fluoro- phores.tugraz.at/substance/


8. BioLegend Spectra Analyzer: https://www.biolegend.com/spectra- analyzer


Jason Kilgore jason.kilgore@thermofisher.com I wanted to quickly point out that: “FP Base (fluorescent


proteins only): https://www.fpbase.org/spectra/” is not fluo-


rescent proteins only (there are huge numbers of dyes) and it has some neat microscope setup options to boot. Mike Nelson msnelson@gmail.com


Trouble Seeding Cells on Gold Grids 3DEM Listserver Dear all,


* The viewer works quite well on mobile devices. You can


find the viewer here: https://public.brain.mpg.de/shiny/apps/ SpectraViewer/ and additional information here: https://brain. mpg.de/326043/spectra- viewer. I would be happy to get your feedback (questions, comments, bug reports, feature requests …). Stephan Junek stephan.junek@brain.mpg.de


Tank you for sharing this. I think the additional functionality


of the 2D viewer, and the scatter plot could be helpful, especially for planning some multilabel spectral imaging experiments. Silas Leavesley leavesley@southalabama.edu


in our lab we have grown mammalian cells suc-


cessfully on holey carbon R2/2 200 mesh gold grids (Quantifoil) for some time now. However, a few months ago we started having problems with cells not attaching, spreading, or looking happy on the grid. Mitochondria stained with MitoTracker sometimes looked normal but were oſten smeared or bloated. Tis differed greatly per experiment. We hypothesized that something might be wrong with


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