Download Green intensity experiment

Green intensity experiment
Experimental setup (experiment conducted on
29 April)
535nm
LED
•
•
•
•
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)
X
X
X
X
X
535nm
LED
X
X
X
X
X
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/m2s)
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/m2s, 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 2nd 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…