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153 Eu peak shape

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shelly

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Posts: 5

Joined: Mon Oct 19, 2015 8:10 pm

Post Fri Oct 23, 2015 11:05 pm

153 Eu peak shape

I recently have trouble with the CV of the 153 Eu beads test. I ran both beads test before and after clean the neutralizer, spray chamber, injector, skimmer/sampler. The Tb counts increased from 500K to 900K. But the CV of the Eu153 increased from 11% to 28%. Could someone kindly give me some hint? I used to change the sample line and sample capillary, this worked to decrease the CV number. Could something major goes wrong which I am not aware of? I should not be able to get the Tb counts around 900K if there is the alignment issue. Am I correct?
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 26, 2015 4:19 pm

Re: 153 Eu peak shape

Hi Shelly,

1. Did you realign the XY, etc after cleaning?

2. Did you *replace* any components, or just clean them?

3. Did you save screenshots or FCS files from your Eu bead acquisitions, so we could look at the data?


Mike
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shelly

Participant

Posts: 5

Joined: Mon Oct 19, 2015 8:10 pm

Post Mon Oct 26, 2015 4:51 pm

Re: 153 Eu peak shape

I did autotuning on XY without changing any parts for cleaning. I have the tuning files and the 153 Eu Massspec files. Please see the attachment. Usually, the CV were little high, it will fine if I changed the sample line and capillary. But this time, it did not help much.
Attachments
tuning file_23OCT2015.png
Beadsaftercleaning_23OCT2015.png
Beadsbeforecleaning_23OCT2015.png
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Mon Oct 26, 2015 4:59 pm

Re: 153 Eu peak shape

Hi Shelly,

In addition to Mike's comments, one of the big factors that I find influences the bead cvs is the oxide level (higher oxidation = wider cvs; poorer resolution). The autotuning will typically maximize your Tb159 dual counts, but I will sometimes follow that with a manual tuning and deliberately reduce either the makeup gas or current a little bit, sacrificing signal intensity for lower oxide ratio, which gives better cvs.

Hope that helps,

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 26, 2015 5:44 pm

Re: 153 Eu peak shape

Hi Shelly,

1. Did you acquire for the same amount of time for the two Eu plots? I see that the tighter CV one has half the events of the broader CV one.

2. I think there are a couple things at issue here. Based on the profile of your single-axis histograms (above and to the right of the axes in the bivariate plots), I think you have a broad CV because you actually have two overlapping peaks. Particularly in the Eu151 (top) plot, the peak isn't symmetric: there's a distinct shoulder on the left (low signal) side of the peak.

It's important to remember that there's a functional difference between large CV from a single, symmetric, but broad peak; and a large CV from a combination of more than one poorly resolved peak. The single-but-broad-peak is a problem: you would be losing mass resolution. Broad-because-multiple peaks, on the other hand, can often result from running your sample too concentrated and therefore causing too many overlapping Event clouds.


In short, you can get a large output CV from two separate and distinct problems. The fact that the broader CV sample is from a larger number of bead Events would be consistent with the too-concentrated-sample idea (assuming you acquired for the same length of time, of course).

Mike
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shelly

Participant

Posts: 5

Joined: Mon Oct 19, 2015 8:10 pm

Post Mon Oct 26, 2015 9:21 pm

Re: 153 Eu peak shape

Hi Mike,

Thank you for your note. The answer to the first question is yes. I always use the same acquisition method for beads tests. I cleaned the system thoroughly before I ran the beads test, I do not quite understand what you mean that the concentrated samples were injected before I clean the system. The sample with narrow CV was ran after I finished all samples. The sample with large CV was ran later after I cleaned the front end parts.

Warmly,
Shelly
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 26, 2015 10:52 pm

Re: 153 Eu peak shape

Hi Shelly,

Sorry, I may have been unclear.

Beadsbeforecleaning_23OCT2015.png
Cluster(s) Events: 9927
(Eu151Di)[m] = 922.7
(Eu151Di)[cv] = 11.8%
- single, symmetric peak

Beadsaftercleaning_23OCT2015.png
Cluster(s) Events: 19413
(Eu151Di)[m] = 1663.7
(Eu151Di)[cv] = 28.3%
- broader peak; likely to be two partially merged peaks, with one being a shoulder at lower signal


I think that "Beadsaftercleaning_23OCT2015.png" represents a bead sample that either was actually at a higher concentration, or the cleaning reduced or eliminated a blockage present before the cleaning (thereby increasing the bead transmission efficiency).

Try running more beads, at half the concentration from that used for "Beadsaftercleaning_23OCT2015.png"; if it's simply a concentration effect merging your populations, a more dilute sample should avoid that, and restore your tighter CV. If that doesn't happen, then there may be more of a hardware issue.


Mike
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shelly

Participant

Posts: 5

Joined: Mon Oct 19, 2015 8:10 pm

Post Tue Oct 27, 2015 8:05 pm

Re: 153 Eu peak shape

Hi Mike,

I forgot to attach the tuning file before I cleaned the instrument. Basically, the Tb counts before the cleaning was half of the Tb counts of the tuning after cleaning. I am not sure if this explains the difference of the events number before and after the cleaning. If the problem is due to the hardware, what should I do to track down?

Thanks,
Shelly
Attachments
tuning file_beforecleaning_23OCT2015.png
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shelly

Participant

Posts: 5

Joined: Mon Oct 19, 2015 8:10 pm

Post Mon Nov 09, 2015 8:46 pm

Re: 153 Eu peak shape

Hi Mike,

As you suggested that it might be the hardware issue. I just realigned the whole front end. A magic just came out. Tb counts reached over 1000K, 153 Eu beads reaches almost 2000 with perfect peak shape and the RSD are within 10%.

Thanks,
Shelly

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