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Tuning post Helios Clean

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mdarrington

Participant

Posts: 2

Joined: Mon May 21, 2018 9:26 pm

Post Wed Sep 12, 2018 1:44 am

Tuning post Helios Clean

Does anyone have trouble getting Tb/Tm over 600K after completing a maintenance clean? We disassemble the Helios every five runs and clean/exchange torch assembly, injector, cones, etc. After reassemble we check background and retune the instrument, but it takes quite a few tries to get Tb/Tm over 600k. Are there any special tricks in reassembling?
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Wed Sep 12, 2018 2:18 am

Re: Tuning post Helios Clean

It may be that your skimmer reducer is dirty or old.

What is the current that you're tuning to?

How old is your skimmer-reducer?

good luck,

Greg
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carynv

Participant

Posts: 5

Joined: Tue Feb 14, 2017 12:36 am

Post Wed Sep 12, 2018 2:32 am

Re: Tuning post Helios Clean

We often have this after cleaning or replacement of cones etc. Generally, increasing the detector voltage by 100 or 200 works well to increase TS signal to a point where Tb is above 600k and the mass resolution tuning step passes. Do this while previewing tuning solution and once you have Tb over 600k perform a full tune. The DV will then be adjusted back to a lower range during tuning.

Good luck!
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mdarrington

Participant

Posts: 2

Joined: Mon May 21, 2018 9:26 pm

Post Wed Sep 12, 2018 3:59 pm

Re: Tuning post Helios Clean

Greg,
We have two skimmer-reducers, one skimmer-reducer is 2 years old (original to the instrument) and the other is 1 year old. We run the Helios about once a week. Each time we clean the instrument we swap out the cones. In other words, we take out the dirty cones to clean them and while they are being cleaned, we place in the clean cones from the previous cleaning.
The present current we are tuning to is 8.

Caryn,
Have there been any detrimental effects?
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carynv

Participant

Posts: 5

Joined: Tue Feb 14, 2017 12:36 am

Post Wed Sep 12, 2018 11:48 pm

Re: Tuning post Helios Clean

No detrimental effect as far as I can tell, it was actually recommended by our fluidigm engineer. According to them, when you clean the instrument or replace the cones, the previous tuning settings (especially XY) could be non-optimal and cause the drop in TS signal. Since the instrument needs to pass mass resolution first, before it can optimise XY etc, you can increase the DV (ex -1800 to -1600) just to get the Tb signal above 600K so that it can pass that first step and continue to optimise XY etc. As I mentioned before it does change the DV back close to the original value so as far as I am aware it does not affect detector lifespan.

As an aside, if changing the DV does not increase the TS signal it is usually a fault at the front-end sample introduction site, which can be checked by increasing the Make-up gas. :D

Hopefully this helps as we struggled with this exact problem!

C
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Sep 13, 2018 1:51 am

Re: Tuning post Helios Clean

I think I'm starting to understand this a bit better. So the tuning fails at first, but once you get it to complete the tuning, the signal is ultimately OK; is that right? (Having "grown-up" manually tuning a CyTOF1, I'm still surprised Fluidigm doesn't tune the XY first in their protocol.)

If so, I think Caryn's suggestion should work well. I suspect it will wear the detector a very tiny bit; just be sure you don't turn it up too much, as I don't think the response to a change in DV is linear.

If the issue is just the XY, you can also simply change the XY manually, it's under "control panel" and you can just manually set it to a position that will give you enough signal to proceed with the tuning. You may also find that each set of your parts consistently tunes to a certain XY position, if that's the case, you can just manually set the XY to the position at which you expect that set of parts works best (based on the previous successful tunings that you have done with those parts) before you start the tuning.

best of luck,

Greg
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Sep 13, 2018 3:04 pm

Re: Tuning post Helios Clean

Hi Caryn, Megan, and Greg,

Megan and Caryn: do you have a Tuning protocol that includes the Pre-XY step? That occurs before Mass Calibration...it's rougher than the later fine XY calibration, but it's usually good enough to start if your X and Y are *really* off, like sometimes happens after a clean (like Greg was suggesting).

A "full protocol" including Pre-XY is something that came pre-loaded onto our instruments:
1. Pre XY
2. Mass calibration
3. XY optimization
4. DV calibration
5. Dual calibration
6. Gases/current optimization
7. QC report

The Quick protocol usually just has Mass calibration, DV, and Dual (or sometimes just Mass cal and Dual). And I've even seen a few Full protocols that left out the Pre-XY......but since the Pre XY only takes at most 1 minute and often helps, I'd recommend doing that if you have one.


When we've had tuning difficulties (XY really off, DV really off, or MUG really off) and finally gotten a decent tune, we usually go ahead and run the tuning *again*. That first tune does a pretty good job, but you can often get another 5-10% signal if you do it again: since the tuning is already mostly optimized, it often does a better job the second time around.


Mike
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vtosevski

Contributor

Posts: 44

Joined: Wed Nov 20, 2013 12:50 pm

Location: Zurich, Switzerland

Post Fri Sep 14, 2018 8:29 am

Re: Tuning post Helios Clean

Hi all,

As Mike suggested, making sure Pre-XY is performed takes care of possible gross miss-alignment that might cause mass calibration to fail. That said, in my experience it only really takes minute amounts of analyte to perform the mass calibration and I don't immediately see this step as causing the poor performance later. If the mass calibration isn't proper, it won't continue the tuning and will stop right there. If it passed, it's not likely causing issues downstream.

Of note here, if the tuning fails someplace after mass calibration, no settings *apart from XY* get applied to the instrument. Might be important to keep that in mind as one goes and repeats the tuning multiple times after failure or so...

My policy has always been to manually get the signal looking good (by manually tweaking pretty much everything auto tuning does except for the DV) and only then perform the auto tune. This way one gets a lot of insight into the instrument performance and what could be suboptimal.

Vinko

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