imaging and CellSpace

tonydoss

Hi,
a couple of questions:

Rob Hutchinson at ESI (now ESL) recommended to have one single mass spec file for laser ablation imaging (thus collecting continuously all the ablation lines), rather than one mass spec file per line. The argument is that laser and mass spec are slightly out of sync, so that results in a fuzzier image than if you collect in one file. I'd prefer to have one mass spec file per ablation line (and use CellSpace), but I'm curious what your views are on this?
in the imaging webinar (fantastic work, by the way! thanks!), you mention than CellSpace can reduce experiment time, showing how ablation lines are covering the sample only. That sounds great, but how do you do that? Do you have to manually draw the ablation lines to they only cover the sample? It takes more machine time to analyse a square/rectangle that cover sample and slide around, but at least it's easy to set up (draw one line and duplicate it). I'd love to learn how to set up ablation such that only the sample is ablated.

Thanks
Anthony Dosseto

Bence

Hi Tony,

Regarding collecting all your data into a single file vs a separate file for each scan line: iolite allows you to use both approaches. There are some advantages to each approach. As Rob mentioned, if you are using a mass spectrometer that has timestamp issues, then collecting everything in a single file will reduce the error associated with this. This was a major issue with Agilent files, but if you copy the entire batch folder across (the xxx.b folder), iolite now reads the timestamp from the associated files to get around some of these problems.

However, collecting all your data in a single file does open the possibility that if something happens to that file (e.g. it is somehow corrupted), you lose your entire image. For our setup in our lab, it is simpler to collect everything in one file, and we haven't had any issues with corrupted files (for the last 10 years or so, at least 😀 ).

Regarding setting up scan lines to only cover your sample for CellSpace, how difficult this is will depend on your laser control software. In at least one laser control software, it's as simple as drawing an outline around your sample, and clicking the "Fill with Lines" button. It only takes about a minute or so (depending on how complex your sample shape is). I haven't used the latest version of ActiveView (ESL), and I've never used Chromium (CETAC), so if anyone has had any experience with those, please feel free to share with the rest of us how to set up this sort of experiment.

Cheers,
Bence

tonydoss

thanks, Bence.
I'll give a go again to have individual mass spec files for each scan line. It's been years since I've done that (I always collect in one file - which is a pain to reconstitute an image with some imaging softwares).

I'll check with ESL if AV1 or AV2 offer the "Fill with Lines" feature (I can't see anything like that on either).

I saw in another post you said the laser needs to drive the mass spec, and not the other way around, for CellSpace to work. Is that correct?
I can't see why the mass spec driving the laser could be a problem as long as you can sync laser log and mass spec data files? but wouldn't surprise me if I was missing something.

Bence

The issue with the mass spec driving the laser is that you would need to set up each sample on the mass spec to run for the length of the line. If you have it the other way around, the laser knows the length of each line and the mass spec just follows along.

On some of our experiments, we don't actually have the laser or mass spec triggering. We just set it up to run for 20 hours (or however long our experiment is) and off it goes. Much simpler in some ways.

Cheers,
Bence

tonydoss

Thanks Bence.
Actually, in a way, with the NWR193 and iCAP-Q, the mass spec drives the laser... but the laser determines the mass spec files: on the iCAP-Q, you set up your sample list by importing the laser pattern list. So that way, the mass spec files are set by the laser pattern.
I'll give it a try, but I reckon that should work.

Anthony