Making them stay (completely) still for confocal microscopy.

I have been trying to immobilise worms for microscopy which involves ~40min aryscan to get some cool pictures.

They keep moving. I am using tetramisole hydrochloride to keep them still, but even at 3% (much higher than most people use) they still move a bit, enough to ruin my pictures!

More precisely, I’m putting them on 2% agarose pads, dropping them into ~5ul tetramisole hydrochloride, putting a coverslip on that and sealing it. I have tried leaving them for an hour or two before imaging but it doesn’t help (much).

Anyone made this work?

25mM NaN3 will completely paralyze worms, no twitching.

Sodium azide will indeed totally paralyze worms, but isn’t suitable for the 40 minute time period keligan asks about. I love azide for looking at worms using Nomarski: it paralyzes them as you say, and they recover really well - so long as they’re not on the slide too long. By 40 minutes on azide you’ll be seeing some really messed up worms with huge vacuoles.

I wonder about some of the ts myosin mutants, but I’ve never looked at the strains.

Maybe you could combine 10% agarose pads, 100nm beads and levamisole or tetramisole. Without anaesthetic they still move a bit but adding anaesthetic could be the solution.

Thanks for the help everyone. We’re trying to look at a stress response transcription factor which complicates things, since NaN3 will trigger this, thus ruining everything. For the same reason I’m not supposed to just kill them.

My current solution, which I haven’t attempted yet is to combine cygel (liquid at 4, solid at 20) with a small amount of concentrated tetramisole. The plan is that the combination of physically holding them in the same place and anesthetizing them will at least stop the most ridiculous flopping around.

We have also found a program which can help us realign our z stacks, so small movement might be cancelled out. I’m interested in the 10% pads and 100nm beads, I wasn’t aware that this was a technique I could use, so I have found the paper and can give it a go if the cygel fails, although it seems like a similar principle.


The issue with levamisole is that it causes hyper-contraction, and muscles can remain twitchy. We have had really good luck with muscimol (10mM), which hyperpolarizes and relaxes all of the muscles without twitching. The only issue is that it paralyzes slower, and it does come off over time, maybe not as fast as levamisole, though. It might also be helpful to use the lite-1(ce314) background, if blue light excitation is stimulating your worms.


Just saw this at CSHL, here’s a good way to immobilize C. elegans if you can set the microfluidics up.

Published on Feb 7, 2017
Real-Time movie taken during an immobilization procedure applied to an L2 animal in a microfluidic chamber

From Developmental Cell (2017), Volume 40, Issue 2, p202–214;

Just as an update, we have managed to get the staying still long enough for 20-45 min airyscan using cygel spiked with M9/1% tetramisole hydrochloride.

If anyone is interested in the specifics shoot me a msg