Minor Post-Meeting Tasks

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John Sexton

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Dec 5, 2016, 6:23:54 PM12/5/16
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Howdy all,

Towards the end of today's meeting we were discussing the different cytometers that we have access to, and I wanted to make sure all the cytometers were consistently documented in the cytometers Google spreadsheet. If any undocumented cytometers could be added to that sheet, that would be much appreciated (I'm mostly thinking of David and Ariel at this point). Feel free to offload that to me if you want to just send me whatever documentation you can find for your cytometer (ideally something that resembles the documents attached).

@Brian and Nicholas: are you guys on the same cytometer? (forgive me for struggling to keep this all straight). Brian, I lost the cytometer specs you posted in the chat of the previous meeting, but could you guys check that I described your cytometer correctly in the Google spreadsheet?

Also, if we have the Spherotech cytometer specifications, that would also be useful to document (not sure where that information is, but I also thought I saw that go by in the meeting chat too).

Thanks!

-John
N002_specs.jpg
N002_light_paths.jpg
N003_lasers.jpg
N003_blue_laser_config.jpg
N003_red_violet_laser_config.jpg
N003_filters.jpg

Nicholas DeLateur

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Dec 5, 2016, 6:27:14 PM12/5/16
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I can confirm that Brian and I are on the same cytometer. The google spreadsheet is specs I uploaded directly from the machine so they should be correct. Forgive my confusion earlier. 

Ross, David J. (Fed)

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Dec 6, 2016, 10:44:07 AM12/6/16
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I filled out the light path info for our Attune cytometer in the Google spreadsheet. I didn’t assign instrument or light path UIDs – since I didn’t know if you wanted them in a particular numbering order. I also scanned and attached the light path diagram from the training manual if that is useful (couldn’t find it online anywhere).

 

Thanks-

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Attune light path.pdf

John Sexton

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Dec 6, 2016, 3:22:34 PM12/6/16
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Awesome! Thanks David and Ariel for updating the cytometers spreadsheet.

As postulated at the end of last meeting, it does indeed look like we have 3 different cytometers with 488nm excitation sources and 530/30 emission filters (LP0006, LP0035, LP0048) with E. coli at all 3 sites!

Attempting to establish our expectations:
  • the raw readout in arbitrary units of E. coli of varying fluorescences (achieved via different expression levels of fluorescent reporters) measured under each of those light paths may not necessarily match? due to differences in excitation laser power and internal cytometer electronics (e.g. gain settings which are not universal)?
  • we expect that calibrating the E. coli fluorescence signal against beads to MEFL units should match? (assuming ideal filters and lasers and "perfect" culture technique, etc. etc.)

On top of this, from Sebastian's spectral analysis here, it looks like, at 488nm excitation, the Spherotech beads spectrum, as measured by Spherotech, suffers a detector saturation shoulder right around 508nm and below. If we could measure a second emission filter set (still with 488nm excitation) which defines a spectral window entirely above this 508nm detector saturation threshold (and not 530/30), we could test our spectral compensation approach (at least with a common excitation source; baby steps).

Candidate light paths: LP0003, LP0017, LP0018, LP0019, LP0020, LP0036, LP0037, LP0049, LP0050.

Not sure we'll get much fluorophore signal out at these long-wavelength light paths (I'm assuming we're sticking with GFP for now): LP0003, LP0018, LP0019, LP0037, LP0050

These light paths may be worth measuring, though?: LP0017, LP0020, LP0036, LP0049

Two of those paths (LP0017, LP0020) look like me and Sebastian, the other two (LP0036, LP0049) look like the "BL2" channel for David and Ariel.

-John

Ariel Haim Hecht

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Dec 6, 2016, 4:39:06 PM12/6/16
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Thanks John. A couple of questions (my apologies in case these questions have already been discussed before I joined the group, I’m still trying to get myself up to speed here):

1) What do you mean by a detector saturation shoulder? Could that be mitigated by reducing the gain?

2) Looking at the wtGFP emission spectrum, there’s not a ton of signal around 590 nm. Do we think we’ll get enough of a signal to be worth analyzing?

3) When you list the two expectations, are those questions to the group or a statement of hypothesis in the form of a question? If they are hypotheses, I think it might help to state them more clearly (and in the form of a declarative statement), because in trying to read them (and having missed the tail end of the discussion yesterday) they feel a little unclear to me.

Thanks!
Ariel


John Sexton

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Dec 6, 2016, 5:16:06 PM12/6/16
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On Tue, Dec 6, 2016 at 3:39 PM Ariel Haim Hecht <arih...@stanford.edu> wrote:
Thanks John. A couple of questions (my apologies in case these questions have already been discussed before I joined the group, I’m still trying to get myself up to speed here):

1) What do you mean by a detector saturation shoulder? Could that be mitigated by reducing the gain?

No worries. This requires an explanation of the spectral compensation strategy, which I don't have time to delve into in depth at the moment (someone else feel free to jump in, though).

Sebastian did a good (albeit sparsely explained) walk-through in a Jupyter notebook in the `test_data\Sebastians Data\Spectra` folder. I've saved that Jupyter notebook file as an HTML file (spectra_analysis.html) in that folder so you shouldn't need to boot up a Jupyter notebook to see it.

I'll draw your attention to plots #4 and #7. Our technique hinges on integrating the bead spectrum, masked by the appropriate filter set window for a given light path, and taking a ratio of those integrals. Unfortunately, the technique didn't work very well with our initial attempt (25% error there at the bottom of that file), and our leading hypothesis is that the integrals of plots #4 and #7 are being contaminated by detector saturation, visualized in those plots as the sharp increase in signal from wavelengths 500nm -> ~508nm.
 

2) Looking at the wtGFP emission spectrum, there’s not a ton of signal around 590 nm. Do we think we’ll get enough of a signal to be worth analyzing?

I'm not sure. It's pretty easy to measure a sample on different channels, though, as long as you know ahead of time, so I was favoring that we measure and see.
 

3) When you list the two expectations, are those questions to the group or a statement of hypothesis in the form of a question? If they are hypotheses, I think it might help to state them more clearly (and in the form of a declarative statement), because in trying to read them (and having missed the tail end of the discussion yesterday) they feel a little unclear to me.


I was mostly just chalking it up to the group for discussion.
 

Ariel Haim Hecht

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Dec 6, 2016, 6:26:27 PM12/6/16
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Where do the data in “spherotech-spectra.csv” come from? Is that provided by spherotech or was it collected in one of our labs?

Jake Beal

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Dec 6, 2016, 7:55:21 PM12/6/16
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Provided by SpheroTech.

Thanks,
-Jake
(sent from my phone)

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