High-Res Bead Spectrum Data

[John Sexton]

Howdy all,

The higher resolution bead spectrum data is now uploaded to the Google Drive folder:

https://drive.google.com/open?id=0Bw3lhsLiX_CvODA1cm8wYUVQQWM

Wrestling with the Tecan software ended up causing a bit of a mess (data is strewn across 4 files), but I've tried to describe everything in the README.md file.

If someone would like to post-process / analyze, by all means be my guest (I left some minor suggestions in the README.md file). Otherwise I'll try and take a look at it when I get a chance.

-John

[jakebeal]

Thanks, John: I just got a chance to look at these.  It's clear that we're not getting as high quality data as we'll need eventually --- the "overflow" region is too large, and probably speaks to inability to narrow the spectrum as much as we'd like, but I think this should be good enough to use as a point of comparison for the data we've got from SpheroTech to make a stopgap until Lili's precision ERF quantification services are available, which looks like to happen around October.

I've added the reference on those, which I just got from John Elliott, into the drive at: https://drive.google.com/open?id=0B4yKj6eAjLFMYVFoQnBGTkVOS2c

Yhanks,

-Jake

[Sebastián Castillo Hair]

I did some basic analysis on John's bead data and produced the following plot:

This is showing the emission intensity (color, "z" axis) as a function of the wavelength (x axis), for different excitation wavelengths (y axis). If you fix the excitation wavelength and plot the intensity vs. emission wavelength (i.e. take a horizontal slice), you have a regular emission spectra as is commonly reported. The intensity numbers were calculated by subtracting the fluorescence measured from a well with a beads suspension, from the fluorescence obtained from a well with just water. White regions are where numbers were not available or the fluorescence measurement was saturated. The black line is the region where the excitation wavelength is equal to the emission wavelength. The analysis was performed in a python notebook (test_data\20160802_bead_spectrum\analysis.ipynb), and the processed data was saved in an excel file (beads_emission_map.xlsx) so we can use it in the future if needed.

Some things that I want to point out:

- As the excitation wavelength is varied, something about the excitation source must remain constant (I assume). It would be nice to know if excitation power (W/m^2) or number of photons (umol/m^2/s of photons) is maintained, or something else, or nothing at all.

- The files that come out of our plate reader specify an emission bandwidth of 20nm and an excitation bandwidth of 10nm, which may not be precise enough for what we need.

- We can see several clumps of intensity in this graph. In particular, as the excitation wavelength changes, we can see that these clusters appear and disappear, consistent with a non-monotonic excitation spectra for each one of the fluorophores inside the beads.

Hope this is interesting.

Sebastian.