Alginate gelling is induced by calcium ions which crosslink gelling polymers at room temperature. To get a homogenous gel, calcium ions cannot be mixed with an Alginate solution but must diffuse from an underlying calcium source. Since the optical properties of Alginate depend on the amount of crosslinking, they ultimately depend on calcium diffusion which is a difficult parameter to control. It is important to note that keeping the same diffusion conditions (time, temperature, calcium source concentration etc.) is necessary to maintain identical alginate optical properties between experiments. In addition to the gelling agent, the nutrient source also impacts light absorbance. Conveniently, LB shows less absorbance than M9 media at most visible wavelengths and was therefore added to our gels for our study.

Data Acquisition Stratgy

We initially attempted to extract the intensity landscape in the gel from a simple picture. The strong diffusion of light from the main laser beam observed on these pictures suggested that an important scattering was occurring in the gel.

Since the scattering observed on gel pictures was questioning the viability of Medusa, we further studied the light landscape with a different strategy. Briefly, we captured the light diffusion at various gels positions on the X-axis (X-axis represents gel depth) by scanning the light intensity along the Z-direction of the gel.
Data Acquisition Strategy Used in this Study

In The Gel The Beam Diameter Is Close to Its Original Value (black)

In addition, to scattering, the laser beam is also affected by absorbance when photons energy is absorbed by gel particles and converted into heat. As expected, absorbance caused the laser intensity to followed a logarithmic decay. We modeled this trend by rearranging Beer-Lambert’s law with the equation defining absorbance:

A(x)= εCx

and

A=Log(I₀/I) (See parameters in Table 2)

In contrast to scattering, the absorbance was high inducing a rapid loss of intensity in the gels. In all gels, the blue laser was more adsorbed than the red. For example, the red laser could reach almost 19.4mm before losing half its initial intensity in Alginate while the blue laser only reached 6.8mm in the same medium. In addition, Agar was the worst performing gel. For example, the blue laser lost half its initial intensity at an Agar depth of only 3.2mm. The absorbance being high, it is necessary to adapt the laser intensity to the gel depth targeted to obtain the right intensity at this depth which will activate the bacteria without bleaching their photoreceptors.

We can extrapolate from the scattering and absorbance fits a light intensity landscape for each gel. This data can be used to predict the light intensity within the direct path and around the laser beam in all positions of the gel volume. Red Laser Profile In 1% Agar

Data validation

Although our light intensity recording strategy was simple, we confirmed the accuracy of our data by comparing the absorbance predicted by our fit at a gel depth of 10mm to the absorbance reading obtained with a cuvette of 10mm path length. We found that the absorbance predicted by our fits matched remarkably well the spectrophotometer absorbance measures for all gels but Alginate (R²=0.77 with Alginate,R²=0.94 without Alginate) . This is probably because of the shape difference between a cuvette the gel mold used in our laser study. The different shapes allowed for different calcium ion diffusion rate which impacted the degree of Alginate crosslinking and, ultimately, its absorbance.