In our meeting with Prof. van der Meer and Drs. Frederic Truffer and Alessandro Crespi, we received critical input on our current prototype to try to get a fluorescence signal above the background noise.
In summary, all the measurements so far are insensitive to arsenic concentration (as compared to the fluorescent plate reader). So we took the bacteria from the vials we use for our prototype (vs the 96 well plate we use for the fluorescent plate reader), and took some volumes to read on the plate reader. We confirmed that the bacteria were fluorescing depending on the arsenic concentration.
Finally, when we plotted samples with two different concentrations of arsenic against time of incubation, we didn’t see any difference between the arsenic concentration samples – rather, they were right on top of each other, both in fluorescence, and in absorbance. But the fluorescence increased over time.
This made us conclude that what we are measuring is the scattering of the incident blue LED light. More bacteria, more scattering, hence the increase in fluorescence over time. We are not measuring the “signal” coming from the arsenic reporting!
This was disappointing, but our meeting gave us plenty of avenues to try for trouble-shooting the signal to noise ratio. Here is the summarized list:
Intensity and wavelength of the excitation light
• the plastic lenses – does it absorb the light?
• test the spectra of the blue LED (not always what the technical sheet says)
• modulate the power of the LED (too much light?)
• reduce scatter – lyse the bacteria to remove particles
• separation of the excitation and the emission wavelengths
– use a new reporter protein LSS mOrange
– use a filter (light gels, RGB filter, commercial filters for microscopy)
– is it sufficiently sensitive?
– is the sensor saturated?
Also, for the transmittance,
• are we detecting transmittance? check
We are going to be working on all of the aspects.