NetNotes


Bob Price University of South Carolina School of Medicine Bob.Price@uscmed.sc.edu


Selected postings are from recent discussion threads included in the


Microscopy (http://www.microscopy.com), Confocal Microscopy (https:// lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy), and 3DEM (https:// mail.ncmir.ucsd.edu/mailman/listinfo/3dem) listservers. Postings may have been edited to conserve space or for clarity. Complete listings and subscription information can be found at the above websites.


Spectral Detector Calibration Confocal Listserver We just had our annual PM visit for our LSM710 and they recalibrated


the 32-channel PMT array. We have several people doing spectral imaging including some people doing 7 color imaging. Tey will need to repeat all of their reference spectra due to the recalibration. Teir new data will be unmixed with different reference spectra relative to data collected before the PM visit. Tis has happened to us several times in the past. One time the users actually asked to go back to the old calibration settings so they could finish out their study. I was just wondering how other facilities handle this complex issue. Do you inform your users that a PM was done, and the instrument calibration has changed? Have people thought about this in terms of reproducibility? Every instrument is different. I would love to hear people’s thoughts. Claire Brown claire.brown@mcgill.ca


Tat’s so frustrating! We have had the same experience many times


with laser intensities: company XXX comes for a PM and as they leave they proudly announce that they have tweaked the alignment and XXX laser line is now, say, 25% or 50% higher intensity compared to yesterday. We have learned that we have to measure laser powers before and aſter a PM, and then we post the old and new numbers right on the booking calendar so that users can scale their data. Usually we remember to do this. I don’t know if I’ve ever mentioned this before (!), but I think it’s pathetic that laser powers are completely uncalibrated on confocal microscopes. Ok, maybe I mentioned it before. In your case, if you have reference spectra from before and aſter the calibration you could create a transformation between the two cases. Perhaps the calibration files that Zeiss produces can be used for this? I don’t know if they are readable with just a text editor. James Jonkman jonkman@uhnresearch.ca


Tat the 32-channel detector is changed could have implications


for AiryScan and AiryScan2 performance over time, since the same 32-channel detector type is used for these (I suppose the field service engineer could be adjusting exactly where the spectrum is being dispersed across the detector). Hopefully the customer has access to the Airy’s raw data. On the Zeiss 510 META 32-channel detector (generation before 710 version of Quasar), the Zeiss field service engineer could -- and occasionally did -- adjust the offset (and maybe the gain) of EACH element independently. I do not know whether Quasar has the same feature. George McNamara geomcnamara@earthlink.net


Changes will be even worse with the Airyscan, as there is one particular channel that always gets the most light (the one in the


76 doi:10.1017/S1551929521000596


middle). Te question is whether in Claire’s case they’re correcting for wavelength driſt (unrelated to the detector), or the detector response itself. Either way, the GaAsP will age unevenly, which won’t really matter in the case of the spectral detector (just don’t use the calibration spectra acquired four years ago), but it will throw off the math behind Airyscan. It should be possible to devise an experiment to test this issue, maybe something like “Pinhole wide open, uniformly fluorescent sample, perhaps way out of focus…” and compare the new calibration with the old one. Best, Zdenek Svindrych zdedenn@gmail.com


I hadn’t previously considered the stability of the multi-anode


array in the Airyscan before; thank you for bringing this to my attention. Our multi-anode systems for spectral detection do clearly driſt over time, with the gain of the individual elements driſting differently over time. We calibrate this periodically with a stabilized tungsten lamp, but in the case of the Airyscan the information is spatial and not spectral. I suppose one could periodically measure a sub-resolution bead, but it would have to be a very consistent sample. I find the various companies that have implemented these multi-anode array PMTs have not given much thought to the stability over time. Te new GaAsP systems will also age more rapidly than “classic” PMTs so I believe the newer, more sensitive arrays will require more monitoring over time. I would be interested in how the vendors actually field recalibrate these spatial systems. Craig Brideau craig.brideau@gmail.com


One of the labs using a confocal here checks the power of lasers


and AOTF output at sample at the beginning of every imaging session to make sure same power is used for all experiments. Tis, however, does not guarantee the same sensitivity of detectors over time and has been an issue when we have had detectors replaced. I don’t know whether they use dyes at known concentrations for calibration. Somewhere I have uranyl glass for green fluorescence, but nobody has used this as a standard for over 7 years. Fluorescent Plexiglas could probably be used as a similar standard (with a few caveats). Michael Cammer michael.cammer@nyulangone.org


We have used green fluorescent glass and fluorescent plexiglass


as well and they are nice because they are very stable. We have also tried measuring the output of a NIST-traceable lamp by placing it at the microscope stage—just be careful with the alignment if you try this. Silas Leavesley leavesley@southalabama.edu


Just to play devil’s advocate, data critical calibrations should be


verified at a set interval, independent of system recalibrations. Some systems parameters can vary with humidity, temperature, debris on the objective lens, simple use, or a previous user may have put the system into an odd configuration. Regularly re-testing the state of the system helps to ensure time is not wasted chasing aſter false results. Even if it is something as simple as re-imaging the same reference slide, or Tetraspeck beads, or any other reference of your choice, this can


www.microscopy-today.com • 2021 May


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92