20230616F section added:

Multiplex multicolor, extremely high FRET efficiency by close proximity (and dipole orientations) of FP chromophore and HaloTag ligand (HTL) ... also some NanoLuc:furimazine based BRET with HT:HTL.

Hellweg 2023 NCB - biosensors XFP-HaloTag high FRET BRET when on - general method development multicolor biosensors with large dynamic ranges Frei Johnsson Hiblot FLIM BLI
https://www.nature.com/articles/s41589-023-01350-1
Here, we introduce a family of designed Forster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence {NanoLuc and Furimazine}. 
* supplemental file(s) at https://www.nature.com/articles/s41589-023-01350-1#Sec43

20230616F section added:

IBEX Imaging Community web portal ... IBEX is (re)iterative multiplex immunofluorescence from Ron Germain's lab, NIH.

Leeson 20230608 addgene blog - IBEX Knowledge-Base - Data Resource for Multiplex Tissue Imaging - re Radtke 2022 NProt Germain Ce3D LiBH4
https://blog.addgene.org/ibex-knowledge-base
https://ibeximagingcommunity.github.io/ibex_imaging_knowledge_base

https://ibeximagingcommunity.github.io/ibex_imaging_knowledge_base/
https://ibeximagingcommunity.github.io/ibex_imaging_knowledge_base/reagent_resources.html

20230614W - listerv post below is self explanatory (other than my mentioning probably an HDD, with SATA-6 SSD, NVMe M.2 drives possibly benefiting too)

From: Confocal Microscopy List <CONFOCALMICROSCOPY@LISTS.UMN.EDU>
Sent: Jun 10, 2023 1:58 PM
Subject: Re: Zeiss Microscope became extremely slow.
Hi everybody. At last I came from a conference and was able to play with the microscope.
Look like chkdsk /f /r fixed the problem.
I shall find something to check the drives more thoroughly. Best regards. Petro.

* GM note1: may work on all drives (HDD, SATA-6 SSDs, NVMe M.2 SSDs, RAID arrays), or might simply be effective for HDDs.

* GM note 2: reminder - defragmentation is only for HDDs (simple HDDs), not SSDs or RAID arrays of any type.

* GM note 3: alsu sueful to run Glary Disk cleaner (ort similar software - I use an old version, circa 2016, that John Gibas, my predecessor downloaded and liked because it did not also install anoying additional software). I also install Glary Undelete on Windows PCs - hopefully will never need this Glary Undelete, but best to have in advance.

20230120F

Immunofluorescence microscopy best practices (in GM's opinion) - 20230120F email ... for JHU colleagues

Assumes: PFA fixed cells or tissue sections on slide … image on confocal (Leica SP8, Olympus FV3000RS), FISHscope (or other widefield microscopes) etc. Recently purchased primary and secondary antibodies, aliquoted and stored correctly (if older antibodies: verify working well on small scale test before spending a lot of time and money on big experiments).

Consider using “Glycine quench” (re: Varsha Singh, G.I.: 100 mM glycine, 30 minutes [gm note: see literature for pH and/or do expt and report optimal pH … may also act as “antigen retrieval”]) to “quench” paraformaldehyde “dangling” from lysines (some expts may in turn be impacted by use of glycine). If using glutaraldehyde (“glut”) or “glut:PFA” mixture, very likely need glycine quench for the “glut”.
Primary antibodies … optimize.
SEVEN fast, large volume washes (per Richard Burry), example fast transfers between Coplin jars or cells in imaging dishes (Mattek glass bottom dishes or WPI Inc … Bin Wu likes less expensive CellView but may have higher fluorescence).
Secondary antibodies … optimize. Can include DAPI (Hoechst 33258 or 33342 also works, more commonly usedfor live cells) in with secondary Ab’s (or with primary if direct label Abs).
SEVEN fast, large volume washes (per Richard Burry for primary antibodies).
Prolong Glass (no DAPI) is best for oil immersion objective lenses (or dry lenses. Prolong Diamond or Prolong Gold ok. Check for air bubbles (by eye or low mag usually enough) and if a problem, gently slide off coverglass, wash off Prolong, apply new Prolong (and never shake the Prolong bottle, and consider firing anyone who does).
Critical: Curing step - Prolong Glass/Diamond/Gold requires TWO or MORE days to solidify (longer if larger coverglass). If using in imaging dish, be sure to replace wash buffer with Prolong (i.e. decant out the wash buffer) and do not “cap” no coverglass on top, leave lid off during 2 day ‘curing”.
NEVER seal the coverglass - Prolong – slide since (i) minor outgassing may continue over longer time (i.e. volatile compound may continue to diffuse out), and (ii) you can REMOVE the coverglass and Prolong by “aqueous soak” (if no urgency, wait one day … if urgent, wait 30 minutes and try to gently slide coverglass off).
Imaging: GM typically uses 20x/0.75NA objective lens, XY pixel size 120nm and Z step 360 nm (confocal) [confocal 1 Airy unit in focus is ~2 um]; 60x/1.4 or 63x/1.4NA objective lens XY pixel size 50nm and Z-step 150nm [confocal 1 Airy unit in focus is 600nm], and typically focus to “middle” of tissue section (i.e. nominal 4 um tissue section from microtome).

Potential refinement: quantitation vs F-actin (or total actin, total tubulin, histone, etc): Consider labeling with Alexa Fluor 647-phalloidin or AF647-anti-actin as an additional channel, and using the pixel and region(s) of interest to quantify in comparison to your experimental channels (i.e. AF555 or AF568 “orange”; AF488 “green”, DAPI DNA counterstain … do not assume that DAPI intensities are quantitative). Analogy is to using (total) “Actin” band as loading control in “quantitative” Western blots (some researchers think that “quantitative western blot” is an oxymoron).

More tips:

“1 Day” = 24 hours (or longer) for this email write-up. For formal protocols and manuscripts, I recommend lab protocol time in hours (or minutes if under 1 hour). One reason: the term overnight is sloppy and varies with latitude (equator vs Baltimore summer and winter vs poles 6 months each).

Whyte 2022 CurrProt “do more with less” … lower concentration primary antibody can result in more binding (example 1:100 for 15 min vs 1:1000 for 24 hours … did not test 48 hours or longer). Direct label antibodies in flow cytometry. May also apply to concentration and staining time for secondary antibodies.

Richard Burry 2010 Immunocytochemistry textbook: 7 washes, keep specimen submerged all the time [1 second transfer to new coplin jar should be ok]  (GM has eBook and one page PDF “flyer”).

GM is a big fan of Brilliant Violet (BV421, BV570, etc) direct label antibodies (BD Biosciences; BioLegend) and similar reagents (Brilliant Ultraviolets for microscopes like FISHscope with ~360nm light source; SuperBrights, more).

GM encourages buying new secondary antibodies.

GM suggests if buying new secondary antibodies (and if “zoo” of species combinations ok) to purchase “Alexa Fluor Plus” (AF488, AF555, AF647 available, anti-mouse, anti-rabbit), 3x brighter due to better linker, ~1.x more expansive than equivalent “standard AF” 2ndAb.

GM is a big fan of tyramide signal amplification (ThermoFisher Alexa Fluor antibodies kits; Akoya Biosciences “Opal”, Bio-Techne/Tocris, Biotium, etc). Almost always should use lower concentration primary antibodies (saving money) compared to standard primary + labeled secondary antibodies. GM recommends PeroxAbolish (BioCare Medical) for “killing” HRP between rounds when multiplexing (Takahashi … Ichii 2012 Cell Transplant. DOI: http://dx.doi.org/10.3727/096368911X586747 ... were at UMiami with GM at the time).

“Glycine quench” (pH 2? 30 min?) may also be useful to “strip” antibodies for iterative label-image-unlabel-repeat multiplexing. There are many multiplex methods out there, a few examples: CycIF, IBEX (Ron Germain, NIH), Akoya CODEX, ChipCytometry/Zellkraftwerk. There are also many ways to destroy fluorophores between rounds (see IBEX papers for some methods, and a few fluorophores resistant, enabling fiduciary markers between rounds).

Think ahead: imaging dishes should have unique labels with lid matching dish, since there is always a risk of confusing lids.

Antibodies go bad over time (depends on antibody, storage details … be aware sometimes tubes get left out overnight or over weekend and then put back in refrigerator … “Trust, but verify” (aka, don’t trust) your lab mates.

Douglas Macarthur: “old soldiers never die, they just fade away” (DM is long dead, so first part is wrong).

GM: “Old antibodies die, please throw them away”.