NetNotes
Propidium iodide (PI) and To-Pro-3 have few ‘details’. Broad- emission spectra should be spectrally (and FLIM) unmixable.
I
encourage our users to use Zombie NIR (BioLegend) both because it is way out in the near infrared and has a cool name. PicoGreen is also very interesting because it can be used at low concentration and ‘lights up’ mitochondrial nucleoids (in addition to nuclei) in live cells. A 2021 Nature Communications article (
https://doi.org/10.1038/s41467- 021-23019-w) published a new dye that they like over PicoGreen (synthesized by their lab). Anti-histone (or antibody to mCherry est for FP-histone H2B) is also a possibility. Segmentation: no DNA counterstain will solve the problem of closely apposed nuclei. I suggest ditching the DNA counterstain and moving to a nuclear pore complex (NPC) or nuclear lamina label. NPCs also provide discrete structures that either work as point-spread function targets or as resolution (or precision localization) test targets. It also helps to multiplex. Tere is no need to be stuck on DAPI, green, red, or NIR. For example, the Germain lab 2020 PNAS published a 65plex sample (up to 11plex per iteration)
https://doi.org/10.1073/pnas.2018488117. George McNamara
geomcnamara@earthlink.net
I second the recommendation of using a nuclear periphery stain
for segmentation. If you’re working with metazoan cells, laminA antibodies give a smoother signal than nuclear pores but either can be useful. Abby Dernburg
afdernburg@berkeley.edu
We like to use laminA antibodies. Tey provide robust staining and are easy to segment. Sylvie Le Guyader
sylvie.le.guyader@
ki.se
Preservation of TdTomato Signal during Tissue Clearing Confocal Listserver We tried ethanol/ECi-based tissue clearing on whole mouse brains
with endogenous TdTomato fluorescent signal. Aſter the clearing, there was no TdTomato-specific signal leſt, but high autofluorescent background was present in the red fluorescent protein range. ECi cleared brain had some yellow color but was clear. Does anyone have experience with using ECi-based tissue clearing methods to clear mouse brain with endogenous TdTomato signals? Or any recommendations on which clearing methods will preserve endogenous TdTomato signal? In addition, what is the best method to reduce the autofluorescent background? Any suggestions would be greatly appreciated. Best, Tingting Gu
tingting.gu-1@
ou.edu
Most oſten it is the dehydration step that causes the loss of
fluorescence. Fluorescent proteins (FPs) require water molecules to maintain their confirmation. Once these are removed, the chromophore falls apart and fluorescence is lost. Methanol is the worst for this, the emission will be lost almost immediately. Ethanol is a little better and THF will give you a day or two of weak fluorescence. Te final clearing solution (DBE, ECi, etc.) also plays a role, but the dehydration step does the most damage. Te most recent solvent-based clearing protocols have found some tricks to better protect the FPs and preserve fluorescence for weeks or months. Tese include: 1) dehydration with 1-propanol or tert-butanol (or a mixture of both); 2) making sure all solutions have a basic pH (∼9.0); 3) keeping the sample at 4o
C during clearing; and 4)
supplementing the solutions with Quadrol or Polyethylene Glycol. For more info see the following: uDISCO Pan et al., Nature Methods, 2016; a-uDISCO Li et al., Frontiers in Neuroanatomy, 2018; and PEGASOS Cell Research, 2018. Doug Richardson
ds.richardson@
gmail.com
We’ve maintained tdTomato expression in brain samples cleared using CLARITY at the Wyss center in Geneva. Te signal is very
2021 September •
www.microscopy-today.com
bright aſter clearing.
https://imgur.com/a/eNbqVEz;
https://i.imgur. com/
ewweZex.png?f]<
https://imgur.com/a/eNbqVEz; TdTomato CLARITY cleared mouse brain
https://imgur.com/a/eNbqVEz. Sverre Grødem
groedem.sverre@
outlook.com
I second the advice from Doug and Sverre and I would also note
that it is important to have a strong promoter with high expression if you want to preserve endogenous expression for clearing. Some protein loss is inevitable, so if you’re starting with low levels and have high autofluorescence (which is common), things will be even harder. Most publications I’ve seen with good retention seem to use Ty-1 or a viral promoter. We have recently published some guidance for navigating these common difficulties with clearing and imaging experiments in a Nature Protocols tutorial-style review:
https://doi.org/10.1038/s41596- 021-00502-8. Since there are so many individual factors that go into any such experiment, we aim to give general advice and warnings, similar to the recent Jonkman confocal tutorial. Among other topics, we include notes on autofluorescence and why the yellowish hue in samples is evidence of protein retention (a good thing!) that shouldn’t interfere with imaging. Hopefully you and others will find it helpful. Good luck! Kurt Weiss
kweiss@morgridge.org
Immersion Fluid Options for Long Time-Lapse Imaging with Long Working Distance Water Objective Confocal Listserver I am demoing a long working distance water objective from Leica (HC
FLUOTAR L 25x/0,95 W VISIR, WD: 2.4mm #506375) in an inverted configuration and am running into some challenges. While the objective is
69
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
