Green intensity experiment

Experimental setup (experiment conducted on 29 April) On an agarose plate, I placed two columns of cells at 5 distances away from a LED Each column (biological repeat) has a single LED (centered at 535nm) illuminating down the plate. On Day 1 (~24 hours after starting the experiment), I took 10-min microscopy time-lapse videos (1 sec/frame) of cells at 3 different regions on each drop : front, center, back In the next few slides I will show the bias and speed profile across the 5 different incident intensities, at the 3 different regions – All analysis was taken over a 100-sec interval – I am showing the values for the first 100-sec interval The data is displayed with the left panel corresponding to results from the left column, and the right panel corresponding to the right column In each slide I also wrote the values in each case for a dark control taken at the beginning of the experiment (Day 0) XXXXXXXXXX XXXXXXXXXX 535nm LED

Images of drops over time Time = 0 Time = 23 hrs (when microscopy is taken)

Time = 120 hrs Cells did not move out of the drops even after 5 days, although one can see some accumulation of cells at the front of the first row of cells (experiencing 28 umol photons/m 2 s)

Bias toward light Dark values: Mean ~ 0.01, std dev ~ 0.4

Bias perpendicular to light Dark values: Mean ~ 0.01, std dev ~ 0.4

In both directions, we don’t see much difference between the bias values of cells under the green light intensities tested, versus dark data. I was confounded especially by the data presented at 28 umol photons/m 2 s, because the cells did accumulate at the front after 5 days, although the average bias values were negative (albeit with a large standard deviation) In videos the cells were definitely motile, so I then tried to look at the speeds of the cells (next two slides)

Speed toward light Dark values: Mean ~ 0.02, std dev ~ 0.02

Speed perpendicular to light Dark values: Mean ~ 0.02, std dev ~ 0.02

In both directions, the speeds of the cells were higher than in the dark. I can’t see any clear trend though. However, whether this is a consequence of the presence of green light (the cells are more motile under light in general but just can’t move directionally in green), or a consequence of cells laying down EPS, is unclear. I propose the following follow-up experiments ( in addition to more repeats under green): – Imaging cells after: placing 24 hours in the dark (which might be the most similar to the condition of placing cells under green light, since they cannot photosynthesize under green light). This would allow us to test the 2 nd hypothesis (cells moving more after laying down more EPS over time) Placing 24 hours in overhead light (which may allow us to compare conditions where cells are placed under light conditions that simply don’t induce directed phototaxis)

Past experiments Started on 23 April (results after 2 days) Started on 16 April (results after 3 days)

I can’t really explain why I get different macroscopic behavior across experiments. Everything was done the same (same setup, same LEDs, same protocol) – Except that for the latest experiment (on 29 April), the plate was kept inside the microscope chamber for 2 days, before being moved into the incubator – The past two experiments have all been kept inside the incubator, and only taken out for imaging in the lab – The temperature conditions are all at 30-degrees C though. I also have a humidifier running inside the microscope chamber, and do not notice an appreciable difference in evaporation from the agarose across those three experiments…