Skeletal muscle cells opto-stimulation by intramembrane molecular transducers

Optical stimulation and control of muscle cell contraction opens up a number of interesting applications in hybrid robotic and medicine. Here we show that recently designed molecular phototransducer can be used to stimulate C2C12 skeletal muscle cells, properly grown to exhibit collective behaviour. C2C12 is a skeletal muscle cell line that does not require animal sacrifice Furthermore, it is an ideal cell model for evaluating the phototransducer pacing ability due to its negligible spontaneous activity. We study the stimulation process and analyse the distribution of responses in multinuclear cells, in particular looking at the consistency between stimulus and contraction. Contractions are detected by using an imaging software for object recognition. We find a deterministic response to light stimuli, yet with a certain distribution of erratic behaviour that is quantified and correlated to light intensity or stimulation frequency. Finally, we compare our optical stimulation with electrical stimulation showing advantages of the optical approach, like the reduced cell stress.


S2. Example of image produced by the Matlab vison recognition code. a.)
The region of interest selected by the user.b.)The withe points represent the sub-area of the ROI.The code evaluate the position of each single points and it use it to follow the movement of an object.

S3. Examples of different contraction path. a.) Contraction profile that follow perfectly the light (casa A). b.) The contraction path starts after several seconds (case B). c.) The contraction path stops before the stimulation protocol (case C). d.)
The myotubes start to contract after the beginning of the protocol and stop before its ends (case D). e.)During the stimulation, the myotubes stop for several second and then start again (case E). f.)The myotubes behave too randomly to be categorised in one of the previous classes (case F).

Energy comparison
Our objective was to compare the energy absorbed by the system during the optical stimulation and the electrical stimulation.We know the delivered power density measured at the sample (  ) and the cross section of the molecule ( =2 * 10 −15  2 ).We have also estimated the number of molecules internalized by the cells,  = 10 10  *  −2 . Eq.1 Through equation 1 we evaluate the energy absorb by time unit and area, and knowing that the duration of a pulse was Δt = 200 ± 0.1 ms, we could evaluate the energy absorbed by the highlighted area.
On the other hand, to evaluate the energy absorbed during the electrical stimulation we made some geometric assumptions.First of all, we assume that the field was almost uniform in the region between the two electrodes and in the region promptly below them, were the cells are located.To evaluate the resistance of the thin sheet of cells we used the resistivity of ρ=5.78 ± 1.4 10 −1 , the distance between the two electrodes  = 7 ± 2 * 10 −3 , the height of the myotubes ℎ = 1 * 10 −5  and the length of the electrode  = 2.4 ± 0.1 * 10 −2 .The resulting resistance of the myotubes is equal to: Eq. 2  =  ℎ = 1.2 ± 0.6 * 10 4  The power P can be obtained through the basic following equation:

𝑅
To evaluate the energy we multiply the power by the duration of the electrical stimulus, Δt = 20 ± 0.1 ms.Then we normalize the energy by the area between the two electrodes given by  =  * , that has a value of 1.69 ± 0.5  2 .We could then compare the two energy absorb by the investigated region as the power density of the light increases or the voltage increases.In figure S5 we report the two trend line.

S5. Energy comparison. The graph report the energy absorb by the cell when they are stimulate with light (red line) or with the electric field (black line).
It is visible from the graph that the energy released to obtain the 100% of cells activation is little bit smaller when we look at the data of the optical stimulation, 5.0 ± 0.5 * 10 −6  *  −2   6.4 ± 3.0 * 10 −6  *  −2 .