Revision as of 00:14, 1 November 2017

OPTIC MODEL

Introduction

Medusa aims at optically controlling bacteria immobilized in a gel by targetting them with two intersecting lasers. Two questions are important to address to achieve this goal:

How far can a laser go in a gel?

What resolution can the laser achieve?

We present in this page our study of the optical properties of commonly used culture gels. We show that it is possible to obtain a clean light signal even at gel depths greater than 10cm.

Gel Choice

The optical properties of a gel depend on the gel type, its concentration and the nutrient source.

	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

Table 1: Gel Included in this Study

The optical properties of a gel depend on the gel type, its concentration and the 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 37C by pre-cooling them (to 6-8C) prior to warm incubation.

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 (1). 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.

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 Fitting and Interpretation

The laser is affected within the gel by scattering because photons are redirected after colliding with gel particles. 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). 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 contrast, 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)=I010^{-εC*x}	I(z)=I_max*exp((z-z₀)²/s))
Parameters	I₀: laser intensity before entering the gel ε: extinction coefficient C: concentration of the absorptive medium.	I_max: the intensity maxima for the diffusion halo recorded in the path of the laser z₀: laser beam position (zero) on Z-axis, S: parameter controlling the spread of the distribution

Table2:

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, the laser intensity followed an absorbance induced logarithmic decay which we modeled by rearranging Beer-Lambert’s law with the equation defining absorbance:

A(x)= εCx

and

A=Log(I₀/I)

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.
The data and fitting parameters for all gel tested are available in our summarizing PDF documents:

Fig:

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 R^{2}=0.77 with Alginate,R^{2}=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.

RNA is a light cost nucleotide material in the cell, We aim to recreate RNA agglomerations as formed in mammalian cells with triple repeat disorders, which show liquid phase separation, forming a organelle-like vesicle, where local concentrations of enzymes can be created.

THIRD MODEL

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