Edited by Bob Price University of South Carolina School of Medicine

Editor’s note: With this issue we welcome the addition of

material from the Confocal Listserver (confocalmicroscopy@ to NetNotes. Te Confocal Listserver, much like the Microscopy Listerver, provides a forum for asking questions about specimen preparation, instrumentation, and image pro- cessing. While NetNotes will focus on current topics of inter- est posted on the Confocal Listerver, the archives dating back to November of 1991 are available by creating an account. We thank Dr. Martin Wessendorf, moderator of the Confocal List- server, and members of the list for allowing Microscopy Today and NetNotes to publish posted material. Selected postings are from January 1, 2019 to February 28,

2019. Postings may have been edited to conserve space or for clarity. Complete listings and subscription information can be obtained at and at https://lists.

Confocal Microscopy Listserver cell culture and hanks balanced salt solution autofluorescence We are trying to image live cultured cells in Hanks Balanced

Salt solution without phenol red. It’s widefield imaging with a standard Chroma GFP filter set, and I’m seeing something I’ve never observed before. Tere’s very high background all over the field that quickly fades within a few seconds. If I stop the acqui- sition briefly and resume, the background goes right back up by about 25%. I’ve never seen this before. What could be the source of this in HBSS, or is it something else? Chris O’Connell coconnell@ January 15, 2019 Hi. We’ve seen this a lot and have come to the conclusion

it’s riboflavin. I think it sticks to the glass/plastic on which the cells are growing and briefly fluoresces before bleaching. It’s replenished from the solution, so once the imaging has stopped, the

signal recovers. Using low-riboflavin medium should

resolve the issue. Simon Walker simon.walker@babraham. January 15, 2019 Vendor reply: We’ve seen the autofluorescence from

riboflavin as well. It’s part of what prompted us at Termo Fisher to release our FluoroBrite DMEM and our Live Cell Imaging Solution (a HEPES derivative), which lack the auto- fluorescent components as well as phenol red (which can par- tially quench visible wavelength dyes). Jason Kilgore jason. January 15, 2019 Vendor comment: Te link below, from 2014, supports

Simon’s riboflavin conclusion and how phenol red serves to quench it. Mistakenly people have felt that phenol itself was auto- fluorescent, but instead it is a quencher and thus can disturb over- all fluorescence as well. And, though not written by us, the link

46 doi:10.1017/S1551929519000403

does reference a product our company, Marker Gene, has been offering for over five years to positive reviews: Opti-Klear—no riboflavin or phenol red, and some of what cells need to thrive for imaging sessions as long as 4 hours, without the need for CO2. fluorescence-in-live-cell-imaging/. Mike Ignatius mjignatius72@ January 16, 2019 Tanks for all the input, everyone, regarding riboflavin.

I didn’t prep the samples, but there may be trace amounts of media leſt aſter they added HBSS. Maybe enough to cause what we are seeing so we can troubleshoot it. Chris O’Connell January 17, 2019

Confocal Microscopy Listserver appropriate lubricants for optical components I want to change grease in condenser and binocular tubes

of a Leica DM IRE2 microscope. Te old grease solidified and made those parts stuck. Can anyone recommend a type and/or supplier of grease for such purpose? Tank you. Petro x.piter@ February 3, 2019 I use white lithium grease for this. Available in hardware and

auto parts stores. Phil Oshel February 4, 2019 Dear Phil, Lithium grease was the first thing that came into

my mind. I used to have a big can of Litol for my bike. But then I have read some scary info that microscope grease has to be very special or it will evaporate, leak, and destroy your equipment, so that kind of stopped me from using it. Have you used it for a long time? My guess is you had no problems with it :) My inten- tion was to put some dumping grease there, like Nye A975. Tis is recommended for focusing mechanisms. Maybe it is overkill. Tanks. Petro February 4, 2019 Tere is grease available for vacuum applications that are

very low off-gassing. If you use that stuff you will probably be fine. It is typically used for moving parts inside vacuum cham- bers or other low-pressure environments. Craig Brideau craig. February 4, 2019 Note that for-real vacuum grease is NOT silicone vacuum

grease. Te silicone grease is only suitable for sealing desiccators. Fomblin is probably the best choice of vacuum grease for mov- ing parts. Be aware that any high-vacuum grease is high-viscos- ity and can be “sticky.” Use a very small amount and apply as a *thin* layer. It’s also expensive, but all true high-vacuum greases are. Also, if you’re applying the grease to any polymer or rubber parts, first check it on a bit that doesn’t matter. Te grease may goo up the polymer over time, especially if the part is warmed or heated. Phil Oshel February 5, 2019 Seems to me that Dow silicone high-vacuum grease is a good choice; they recommend it for optical components, • 2019 May

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