Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo

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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/5/51/Oscilloscope.png" alt="Oscillogram" width="650px" /></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/5/51/Oscilloscope.png" alt="Oscillogram" width="650px" /></p>
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                                         <p id="legend">Figure 3.<br>Oscillograms showing the two different mode of the photodiode.</br>The first oscillogramm shows the pulse train mode and the second the 50% duty cycle mode</br></br></p>
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                                         <p id="legend">Figure 3.<br>Oscilloscope recordings showing the two different modes of the photodiode.</br>The first recording shows the pulse train mode and the second the 50% duty cycle mode</br></br></p>
                                         <p>Since this frequency will be calculated by the Arduino controller, it may cause some trouble to the program to use a pulse train because the duration of the pulse is always 500ns and can be missed by the controller. The square wave (50% duty cycle) seems to be a better solution because of the 50% duty cycle. It means that the pulse duration depends on the frequency. Its duration is equal to 1/2f and since the light intensity we want to measure will be low, this type of signal can be easily detected by Arduino.</br></br></p>
                                         <p>Since this frequency will be calculated by the Arduino controller, it may cause some trouble to the program to use a pulse train because the duration of the pulse is always 500ns and can be missed by the controller. The square wave (50% duty cycle) seems to be a better solution because of the 50% duty cycle. It means that the pulse duration depends on the frequency. Its duration is equal to 1/2f and since the light intensity we want to measure will be low, this type of signal can be easily detected by Arduino.</br></br></p>
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<p id="legend">Figure 4.<br>Characterization of the algorithm in Arduino</br>
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<p id="legend">Figure 4.<br>Characterization of the algorithm in Arduino
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The first graph shows us the reponse of Arduino in pulse train mode, the second one shows us the response of Arduino in 50% duty cycle mode, and the last one gives us the standard deviation of the 50% duty cycle mode</br></br></p>
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The first graph displays the reponse of Arduino in pulse train mode, the second one displays the response of Arduino in 50% duty cycle mode, and the last one gives us the standard deviation of the 50% duty cycle mode</br></br></p>
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                                         <p>For the proof of concept of the optical part we use a LED lamp - MR16 (GU5.3)- and a cube filter from a fluorescence microscope with excitation and emission filters and an adjustable lens. The LED lamp was chosen so that we didn't have to buy <strong>high-power LEDS</strong> and build a <strong>card with heat sinks</strong>. This lamp illuminates with <strong>520 lumens in a 40° cone under 12V and 6W</strong>. The low voltage was chosen as <strong>a safety measure</strong> and the small angle to <strong>avoid losing too much light</strong>. The excitation filter is a <strong>green interferential filter</strong> to excite the red fluorescent protein and the <strong>red emission filter</strong> is only a colored filter to collect all the red light in order to have a more precise measure. In the cube there is also a <strong>dichroic mirror</strong> that reflects all the green light and transmits all the red light. This mirror enables us to <strong>separate the photodiode from the light source completely</strong>.</br></br></p>
                                         <p>For the proof of concept of the optical part we use a LED lamp - MR16 (GU5.3)- and a cube filter from a fluorescence microscope with excitation and emission filters and an adjustable lens. The LED lamp was chosen so that we didn't have to buy <strong>high-power LEDS</strong> and build a <strong>card with heat sinks</strong>. This lamp illuminates with <strong>520 lumens in a 40° cone under 12V and 6W</strong>. The low voltage was chosen as <strong>a safety measure</strong> and the small angle to <strong>avoid losing too much light</strong>. The excitation filter is a <strong>green interferential filter</strong> to excite the red fluorescent protein and the <strong>red emission filter</strong> is only a colored filter to collect all the red light in order to have a more precise measure. In the cube there is also a <strong>dichroic mirror</strong> that reflects all the green light and transmits all the red light. This mirror enables us to <strong>separate the photodiode from the light source completely</strong>.</br></br></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/c/ca/Optique.png" alt="Fluorometer_igem2013_Grenoble-EMSE-LSU" width="600px" /></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/c/ca/Optique.png" alt="Fluorometer_igem2013_Grenoble-EMSE-LSU" width="600px" /></p>
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                                         <p id="legend">Figure 5.<br>TALKE'coli: C2M part<br>
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                                         <p id="legend">Figure 5.<br>TalkE'coli: C2M part
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                                        On the left: the real device, on the right: functional schematic<br>
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On the left: the real device, on the right: functional scheme</br>
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                                        The light from the LED lamp goes through the green excitation filter and illuminate the sample thanks to a dichroic mirror. Then the red fluorescent protein is now excited and re-emits red light that goes through a lens that concentrate it on the photodiode.</br></br>  
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The light from the LED lamp goes through the green excitation filter and illuminates the sample thanks to a dichroic mirror. Then the red fluorescent protein is now excited and re-emits red light that goes through a lens that concentrates it on the photodiode.</br></br>  
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<p>With the setup shown above, we put different culture in a 50mL rounded tube and to protect the photodiode from the outside lamp we place all the component in a large box. These are the results we obtained:</br></br></p>
<p>With the setup shown above, we put different culture in a 50mL rounded tube and to protect the photodiode from the outside lamp we place all the component in a large box. These are the results we obtained:</br></br></p>
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<p align="center"></br></br><img src="https://static.igem.org/mediawiki/2013/0/0a/Charac_fluo_measure.png" alt="Charac_fluo_measure" width="600px" /></br></br></p>
<p align="center"></br></br><img src="https://static.igem.org/mediawiki/2013/0/0a/Charac_fluo_measure.png" alt="Charac_fluo_measure" width="600px" /></br></br></p>
<p id="legend">Figure 6.<br>Characterization of the fluorescence measurements</p>
<p id="legend">Figure 6.<br>Characterization of the fluorescence measurements</p>
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<p>Here we can see that there is a linear equation between the Fluorescence measure with the microplate reader Tristar and our setup. That means firstly that our <strong>can detect the red fluorescence of KillerRed</strong> and that we can easily <strong>link its measurement with our model</strong> since it use the fluorescence measured by the Tristar to find all the parameters.
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<p>The fluorescence readings of TalkE'coli and of the Tristar microplate reader are <strong>linearly related</strong>. Furthermore, the precision of both measurements are comparable. Our device is therefore <strong>able to detect KillerRed fluorescence with enough precision</strong> to allow proper cell growth control.
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                                     <h2 id="Electronic">Electronic circuit</h2>
                                     <h2 id="Electronic">Electronic circuit</h2>
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                                       <p>To kill more or less cells, our device needed to control light intensity. To do so, we made the electronic circuit shown below.</br></br>
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                                       <p>The light intensity needs to be adjusted with precision to <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology">control the living cell density</a>. We therefore included the electronic circuit shown below to control light intensity.</br></br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/f/f2/Highpowerled.png" alt="our electronic circuit" width="350px" /></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/f/f2/Highpowerled.png" alt="our electronic circuit" width="350px" /></p>
                                         <p id="legend">Figure 7.<br>Electronic circuit that enables us to control light intensity</br>
                                         <p id="legend">Figure 7.<br>Electronic circuit that enables us to control light intensity</br>
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This circuit stabilizes the amperage of the LED lamp at 0.5A thanks to a bipolar transistor, three diodes and the R3 and R4 resistors.
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This circuit stabilizes the current of the LED lamp at 0.5A thanks to a bipolar transistor, three diodes and the R3 and R4 resistors.
</br>The MOS transistor is controlled by Arduino and is used like a switch. It allows us to control the average light intensity of the LED lamp.</br></br>                                       
</br>The MOS transistor is controlled by Arduino and is used like a switch. It allows us to control the average light intensity of the LED lamp.</br></br>                                       
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<p>The first part of this circuit – all components above the MOS transistor BS170 - stabilizes the amperage of the LED lamp and the second – composed by the MOS transistor and Arduino - allows us to control the average light intensity of the LED lamp.</br>
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<p>The first part of this circuit – all components above the MOS transistor BS170 - <strong>stabilizes the current of the LED lamp</strong> and the second part consisting in the MOS transistor and Arduino microcontroller- allows us to <strong>control the average light intensity</strong>.</br>
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The nominal power of the LED is 6W when 12V is applied. That means that the amperage going through the LED lamp is 0.5A.</br>  
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The nominal power of the LED is 6W when 12V is applied. That means that the current through the LED lamp is 0.5A.</br>  
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Since we cannot be sure that our alimentation is completely stable, we need to stabilize it thank to a bipolar transistor, three diodes and two resistors.</br></br>  
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To ensure that the power supply is stable enough, we stabilized it thanks to a bipolar transistor, three diodes and two resistors.</br></br>  
We know that:</br></br>
We know that:</br></br>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/3/34/Eq_i_transistor.PNG" alt="law intensity transistor" /></br></br></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/3/34/Eq_i_transistor.PNG" alt="law intensity transistor" /></br></br></p>
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<p>
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We are now able to control light intensity thanks to this circuit, but to plug all the electronic parts - the photodiode and this circuit - a Printed circuit board (PCB) need to be printed. To do so, we used the software called Altium.</br></br>
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We are now able to control light intensity thanks to this circuit, but to connect all the electronic parts - the photodiode and this circuit - a Printed circuit board (PCB) was printed, using the Altium software.</br></br>
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We can finally create the box to finish the device.
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We finally designed a box to enclose our device.
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<img src="https://static.igem.org/mediawiki/2013/a/a8/Servo_pos2.png" alt="Position2_servo" width="450px" /></p>
<img src="https://static.igem.org/mediawiki/2013/a/a8/Servo_pos2.png" alt="Position2_servo" width="450px" /></p>
<p id="legend">Figure 9.<br>On the left, the first position of the servomotor and on the right, the second position of the servomotor.</br>
<p id="legend">Figure 9.<br>On the left, the first position of the servomotor and on the right, the second position of the servomotor.</br>
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Known dimensions :</br></p>
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</p>
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<p align="left"><strong>L</strong>: distance between the center of the servomotor S and the center of the hole in the box A (6.5cm)</br>
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<p align="left">Known dimensions :</br>
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<strong>L</strong>: distance between the center of the servomotor S and the center of the hole in the box A (6.5cm)</br>
<strong>h</strong>: height from A to S (2cm)</br>
<strong>h</strong>: height from A to S (2cm)</br>
<strong>R</strong>: radius of the filter and also the hole in the box (1cm)</br>
<strong>R</strong>: radius of the filter and also the hole in the box (1cm)</br>
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                                     <h2 id="Box">Building the box</h2>
                                     <h2 id="Box">Building the box</h2>
                                       <p align="center"><object width="480" height="360"><param name="movie" value="//www.youtube.com/v/qruRM62kY-k?hl=fr_FR&amp;version=3"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/qruRM62kY-k?hl=fr_FR&amp;version=3" type="application/x-shockwave-flash" width="480" height="360" allowscriptaccess="always" allowfullscreen="true"></embed></object></br></br></p>
                                       <p align="center"><object width="480" height="360"><param name="movie" value="//www.youtube.com/v/qruRM62kY-k?hl=fr_FR&amp;version=3"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/qruRM62kY-k?hl=fr_FR&amp;version=3" type="application/x-shockwave-flash" width="480" height="360" allowscriptaccess="always" allowfullscreen="true"></embed></object></br></br></p>
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<p>To build the box, we used the <strong>Computer-Aided Design (CAD) software Google SketchUp</strong>. It allows us to use a <strong>3D-printer</strong>, and <strong>save time</strong> on the construction process. Our device needs to fulfill several specifications. It needs to <strong>be mountable in an incubator</strong>, so that the culture is at 37°C under agitation and <strong>protected from outside light</strong>, but it also has to have enough space to accommodate the electronic circuitry and optical components.</br></br></p>
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<p>To design the box, we used the <strong>Computer-Aided Design (CAD) software Google SketchUp</strong>. It allows us to use a <strong>3D-printer</strong>, and <strong>save time</strong> on the construction process. Our device fulfills several specifications. It needs to <strong>be mountable in an incubator</strong>, so that the culture can <strong>be agitated at 37°C</strong> and <strong>protected from outside light</strong>. Enough room should also be made to accommodate <strong>the electronic circuit and the optical components</strong>.</br></br></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/d/dd/Box_parts.png" alt="box_parts" width="500px"><p id="legend">Figure 10.<br>Main parts of the device</br>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/d/dd/Box_parts.png" alt="box_parts" width="500px"><p id="legend">Figure 10.<br>Main parts of the device</br>
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                                         <strong>1</strong> - The box with two separated parts where there are the electronic circuitry and optical components in one part and the Erlenmeyer with our engineered bacteria in the other.  </br>
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                                         <strong>1</strong> - The box with two separated parts containing (i) the electronic circuit and the optical components and (ii) the Erlenmeyer with our engineered bacteria.  </br>
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                                         <strong>2.a</strong> - The filter rack with, from left to right, the excitation and emission filters and the dichroic mirror, the red colored filter and a plane mirror and only a mirror that reflects the white light from the LED lamp</br>
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                                         <strong>2.a</strong> - The filter rack with, from left to right, the excitation and emission filters and the dichroic mirror to record Killer Red fluorescence, the red colored filter and a plane mirror to induce Killer Red expression and a mirror that reflects the white light from the LED bulb for Killer Red activation.</br>
                                         <strong>2.b</strong> - The rail where the filter rack can slide</br>
                                         <strong>2.b</strong> - The rail where the filter rack can slide</br>
                                         <strong>3</strong> - The lens holder</br>
                                         <strong>3</strong> - The lens holder</br>
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<h3>The box</h3>
<h3>The box</h3>
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<p>The box has been made to be mountable in an incubator. On its support, there are holes that fit the holes of the incubator so that you can simply screw them together. There are also two doors, one with a latch that enables us to easily put the Erlenmeyer and a second dedicated to the electronic circuitry and optical components. As a safety and experimental issue, the two parts are completely separated to avoid spreading the culture on the electronic part or illuminating the culture with an unwanted wavelength. There is only one hole that allows us to illuminate the sample.</p>
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<p>The box has been made to be mountable in an incubator. On its support, there are holes that fit the holes of the incubator so that you can simply screw them together. There are also two doors, one with a latch that enables us to easily put the Erlenmeyer and a second dedicated to the electronic circuitry and optical components. As a safety and experimental issue, the two parts are completely separated to avoid spreading the culture on the electronic part or illuminating the culture with an unwanted wavelength. There is only one window that allows us to illuminate the sample.</p>
<h3>The filter rack and the rail</h3>
<h3>The filter rack and the rail</h3>
<p><img src="https://static.igem.org/mediawiki/2013/f/fb/Talk%27E_filter.png" alt="filter_rack_inside_box" style="float:left;margin: 25px;" width="300px"/>To create the filter rack, we were inspired by a cube filter which is composed of two excitation filters – green and blue, two emission filters – red and yellow, and two dichroic mirrors.  We re-designed it on SketchUp by adding two additional slots. The first slot is used to measure the red fluorescence of KillerRed. There is a green excitation filter on the top, a red emission filter on one side and a dichroic mirror between the two pieces.  A red colored filter is on the top of the second slot to induce the KillerRed protein production. There is no filter in the third slot because it is used to activate ROS emission with white light. Since the light comes from above, there is a plate mirror between the two pieces under the slots two and three. The last slot was planned for further use, for instance to measure back-scattering of the cell suspension. For such measure, a colored filter and a half-reflecting mirror would be used. Back-scattering would provide information about the total number of bacteria, similar to OD600nm recording.</br>
<p><img src="https://static.igem.org/mediawiki/2013/f/fb/Talk%27E_filter.png" alt="filter_rack_inside_box" style="float:left;margin: 25px;" width="300px"/>To create the filter rack, we were inspired by a cube filter which is composed of two excitation filters – green and blue, two emission filters – red and yellow, and two dichroic mirrors.  We re-designed it on SketchUp by adding two additional slots. The first slot is used to measure the red fluorescence of KillerRed. There is a green excitation filter on the top, a red emission filter on one side and a dichroic mirror between the two pieces.  A red colored filter is on the top of the second slot to induce the KillerRed protein production. There is no filter in the third slot because it is used to activate ROS emission with white light. Since the light comes from above, there is a plate mirror between the two pieces under the slots two and three. The last slot was planned for further use, for instance to measure back-scattering of the cell suspension. For such measure, a colored filter and a half-reflecting mirror would be used. Back-scattering would provide information about the total number of bacteria, similar to OD600nm recording.</br>
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<h3>The lens holder</h3>
<h3>The lens holder</h3>
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<p>To focus the beam of the fluorescence, we use a microscope objective. Therefore, we needed a part that could hold it. We created a mold where you just plug the objective into it.</p>
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<p>To focus the beam of the fluorescence, we used a microscope objective, held in place by a tailored mold.</p>
<h3>The LED lamp box</h3>
<h3>The LED lamp box</h3>
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<p>To avoid illuminating the entire box, we put the LED in a smaller one where one side is closed with only a hole that matches the size of the lamp cap. Since the illumination angle of the lamp is small, the light goes almost in one direction and only light up the filters or mirror.</p>
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<p>To avoid illuminating the entire box we inserted the LED in a smaller box with an opening that matches the size of the lamp cap. Since the illumination angle of the lamp is small, the light goes almost in one direction and only lights up the filters or the mirrors.</p>
<h3>The servomotor holder</h3>
<h3>The servomotor holder</h3>
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<p>Since the servomotor will move the filter rack, it needed to be securely attached onto the box. We designed a part with two arms that allowed you to screw the servomotor and a flat support to bold it on the box. We drilled many holes so that we can easily adjust the height of the servomotor.</br></br>
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<p>Since the servomotor will move the filter rack, it needed to be securely attached onto the box. We designed a part with two arms where you can screw the servomotor and a flat support to hold it on the box. We drilled many holes so that we can easily adjust the height of the servomotor.</br></br>
As said previously we used a 3D-printer to built these parts, but because of the complexity of the filter rack and its rail they had to be done with a another 3D-printing method. These two parts were completed through Selective Laser Sintering and all the other were made by Fused Deposition Modeling</p>
As said previously we used a 3D-printer to built these parts, but because of the complexity of the filter rack and its rail they had to be done with a another 3D-printing method. These two parts were completed through Selective Laser Sintering and all the other were made by Fused Deposition Modeling</p>

Latest revision as of 03:10, 5 October 2013

Grenoble-EMSE-LSU, iGEM


Grenoble-EMSE-LSU, iGEM

Retrieved from "http://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Instrumentation/Fluo"