Team:Paris Bettencourt/Hardware setup
GEL OPTICS
Medusa aims at optically controlling bacteria immobilized in a gel with two intersecting lasers. Two questions are important to address to achieve this goal:
In this page, we present our study on the optical properties of three commonly used culture gels. We develop a simple strategy to record and model the light profiles of our lasers and show that a clean light signal can be obtained at significant gel depths (>10cm).
- How far can a laser go in a gel?
- What resolution can the laser achieve?
In this page, we present our study on the optical properties of three commonly used culture gels. We develop a simple strategy to record and model the light profiles of our lasers and show that a clean light signal can be obtained at significant gel depths (>10cm).
Gel & Laser Choice
The optical properties of a gel depends on the gel type, its concentration and the nutrient source.
In addition, the profile of a laser beam in the gel depends on the laser wavelength, beam diameter and intensity.
Characteristics of the lasers used in this study
| Alginate | Agar | Gelrite | |
|---|---|---|---|
| Key advantage | Solidifies at room temperature by adding calcium ion |
Most ubiquitous microbiology gel | Low absorbance |
| Recommended concentration | 1.75-4% | 1-1.5% | 0.15-0.25% |
| Melting-Gelling Point | NA | 0.5%: 85-20C 1%: 90-35C |
0.5%: 65-15 1%: 100-35C |
| Key disadvantage | Properties depend on calcium ion diffusion which is hard to control |
High absorbance | Melts less well than agar |
| Concentration studied | 1% with LB | 1% with LB | 1% & 0.5% with LB |
Gel type, concentration & nutrient source
It is best to use a low gel concentration to reduce absorbance. Conveniently some gels have a higher melting point than solidifying point. This hysteresis phenomenon means that the gel concentration can be lowered and still be kept at in a solid state at 37oC by pre-cooling them (to 6-8oC) prior to warm incubation.
Gels Melt at a Lower Temperature Than They Melt (L: liquid, T: transition, S: solid
Gels Melt at a Lower Temperature Than They Melt (L: liquid, T: transition, S: solid
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
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
Initial Data Acquisition Strategy
Gel Images With Light Halo
Data Fitting and Interpretation
In The Gel The Beam Diameter Is Close to Its Original Value (black) 
The laser is affected within the gel by scattering because photons are redirected after colliding with gel particles.
In addition, scattering causes the laser to diffuse and creates a light halo surrounding the main beam. We found that the intensity of this light halo described a normal distribution on the Z-axis which we fitted a Gaussian equation (See table 2). The spread of this distribution represents the diameter of the laser beam.
In contrast to what the gel pictures suggested, the laser diameter within the gel was actually close to its initial diameter, prior to entering the gel. The large light diffusion observed on gel pictures was probably caused by internal camera corrections which automatically balanced areas of high and low light intensity. In fact, the low scattering that we recorded manually is very encouraging as it shows that a high targeting precision can be obtained within a gel which will give a high resolution to Medusa control.
| Absorption | Scattering | |
|---|---|---|
| Profile |  |
 |
| Trend | Logarithmic decay | Normal distribution |
| Axis of Occurrence | X-axis | Z-axis |
| Equation Fit | I(x)=I0*10-ε*C*x | I(z)=Imax*e((z-z0)2/s)) |
| Parameters |
|
|
Table 2: Scattering and Absorbance Profiles and Fits
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:
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.
A(x)= εCx
andA=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.
Distance At Which The Laser Looses Half Its Initial IntensityWe 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
Red Laser Profile In 1% Agar
Red Laser Profile In 1% Agar
The data and fitting parameters for all gel tested are available in our summarizing PDF documents:
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.
In addition, to the absorbance, we seeked to demonstrate that our prediction of the laser diameter at a given gel depth was also accurate. To do so, we exposed the high resolution light curing resin S-Pro (kindly provided by our industrial sponsor Spot-A Materials) to our laser after passing through a range of gel thicknesses or a range of optical filters of equivalent absorbance . S-pro is a 3D printing resin which cures at our blue laser’s wavelength. We measured the diameter of the resin particles solidified after 1 minute exposure to the blue laser. This gave us a proxy of the laser beam diameter after its path through the gel. Here again, we found that the diameter of the particle solidified (ranging from 2.6 to 1.9mm) was in excellent agreement with the diameter of light the diffusion predicted by our model (diameter 2.0mm).
Altogether, our results show that, despite its simplicity, our data acquisition and fitting strategy could accurately quantify absorption and scattering within our gels.
Altogether, our results show that, despite its simplicity, our data acquisition and fitting strategy could accurately quantify absorption and scattering within our gels.
Conclusions
We have developed a simple yet powerful way of measuring and fitting the light profile of a laser in a gel. Our data, which accuracy was confirmed, show that little light scattering can be obtained. The high absorbance limited the outreach of the laser beam but this can be corrected by using a stronger laser or a lower gel concentrations to reach a depth greater than 10cm.
Together, these results confirm the feasibility of achieving a high targeting resolution at significant length within the gel medium.
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com
bettencourt.igem2017@gmail.com 
