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

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                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/a/a7/Eglometer.png" alt="The Eglometer" width="500px" /></p>
                                         <p align="center"><img src="https://static.igem.org/mediawiki/2013/a/a7/Eglometer.png" alt="The Eglometer" width="500px" /></p>
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                                         <p id="legend"><strong><em>The E. glometer of Cambrige team (iGEM 2010)</em></strong></br>
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                                         <p id="legend">Figure 1.<br>The E. glometer of Cambrige team (iGEM 2010)</br>
                                         Device built by Cambrige team in 2010 to measure the light intensity of their LuxBrick</br>
                                         Device built by Cambrige team in 2010 to measure the light intensity of their LuxBrick</br>
                                         <em><strong>Source:</strong></em><a href="https://2010.igem.org/Team:Cambridge/Tools/Eglometer">https://2010.igem.org/Team:Cambridge/Tools/Eglometer</a></br></br>
                                         <em><strong>Source:</strong></em><a href="https://2010.igem.org/Team:Cambridge/Tools/Eglometer">https://2010.igem.org/Team:Cambridge/Tools/Eglometer</a></br></br>
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                                         <p align="center", style="margin:20px"><img src="https://static.igem.org/mediawiki/2013/5/5f/IGEMerworkphotodiode.png" alt="memberworkingonphotodiode" width="500px"></p>
                                         <p align="center", style="margin:20px"><img src="https://static.igem.org/mediawiki/2013/5/5f/IGEMerworkphotodiode.png" alt="memberworkingonphotodiode" width="500px"></p>
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                                         <p id="legend"><strong><em>A member of the team working on the photodiode</em></strong></br></br></p>
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                                         <p id="legend">Figure 2.<br>A member of the team working on the photodiode</br></br></p>
                                         <p>For the same amount of light, we measure the frequency at the output of the photodiode for a pulse train or a square wave (50% duty cycle). According to the datasheet, when using a pulse train the linear relation between the frequency and the irrandiance is given by 1kHz=1µW/cm². When using a square wave (50% duty cycle) it is 1kHz=2µW/cm². This is what we can see on the figure below.
                                         <p>For the same amount of light, we measure the frequency at the output of the photodiode for a pulse train or a square wave (50% duty cycle). According to the datasheet, when using a pulse train the linear relation between the frequency and the irrandiance is given by 1kHz=1µW/cm². When using a square wave (50% duty cycle) it is 1kHz=2µW/cm². This is what we can see on the figure below.
</br></br></p>
</br></br></p>
                                         <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"><strong><em>Oscillograms showing the two different mode of the photodiode.</em></strong></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>
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                                         <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 depend 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>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>
                                                 <h3>Arduino</h3>
                                                 <h3>Arduino</h3>
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</br></p>
</br></p>
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<p id="legend"><strong><em>Characterization of the algorithm in Arduino</em></strong></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>According our the experience, the pulse train mode is not a good option since the curve doesn’t follow at all the x=y curve. Only three points are shown here because the others are worst. On the other hand, the 50% duty cycle mode seems to work better, at least at the low frequency. For frequencies under 35kHz the curve fits the equation y=x. However above this critical frequency, the response of the microcontroller seems to break down and follows the equation y=x/2. For frequencies over 100kHz, the system does not give reliable results. This is explained by the time of the "while loop" in the microcontroller program. At the end of this loop the program jump back to the beginning of the loop, but when the photodiode emits peaks at increasing frequencies, the microcontroller is not fast enough and misses one pulse out of two which explains the curves y=x then y=x/2. efficient at all, that makes us believe this is the right explanation. In addition, the plot of the standard deviation as a function of the frequency demonstrates that the system is very precise for low light intensities. The errors are below 0.5% when 100 pulses are recorded. At the lowest illuminations, the device will measure the fluorescence of the bacterial culture every 5 min, which is enough for this kind of sample. In the next paragraph, we are going to see that the device is efficient enough to measure low light intensity like fluorescence.
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<p>According to our experiment, the pulse train mode is not a good option since the curve doesn’t follow at all the x=y curve. Only three points are shown here because the others are worse. On the other hand, the 50% duty cycle mode seems to work better, at least at the low frequency. For frequencies under 35kHz, the curve fits the equation y=x. However, above this critical frequency, the response of the microcontroller seems to break down and follows the equation y=x/2. For frequencies over 100kHz, the system does not give reliable results. This is explained by the time of the "while loop" in the microcontroller program. At the end of this loop, the program jumps back to the beginning of the loop, but when the photodiode emits peaks at increasing frequencies, the microcontroller is not fast enough and misses one pulse out of two which explains the curves y=x then y=x/2. In addition, the plot of the standard deviation as a function of the frequency demonstrates that the system is very precise for low light intensities. The errors are below 0.5% when 100 pulses are recorded. At the lowest illuminations, the device will measure the fluorescence of the bacterial culture every 5 min, which is enough for this kind of sample. In the next paragraph, we are going to see that the device is efficient enough to measure low light intensity like fluorescence.
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<h2 id="Fluo">Fluorescence Measurement</h2>
<h2 id="Fluo">Fluorescence Measurement</h2>
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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 high-power LEDS and build a card with heat sinks. This lamp illuminates with 520 lumens in a 40° cone under 12V and 6W. The low voltage was chosen as a safety measure and the small angle to avoid losing too much light. The excitation filter is a green interferential filter to excite the red fluorescent protein and the red emission filter 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 dichroic mirror that reflects all the green light and transmits all the red light. This mirror enables us to separate the photodiode from the light source completely.</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 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"><strong><em>TALKE'coli: C2M part</em></strong><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.  
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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>
</p>
</p>
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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>
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<table align="center" style="border-collapse:collapse;font-size: 14px;">
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  <tr style="border:1px solid black;">
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      <th>Sample</th>
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      <th>Measure by Tristar [RLU]</th>
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      <th>Frequency by the oscilloscope [Hz]</th>
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      <th>Irradiance by the oscilloscope [nW/cm²]</th>
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  </tr>
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  <tr style="border:1px solid black;">
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      <td>LB medium</td>
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      <td>142 +/- 18.3 </td>
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      <td>15.3 +/- 0.4</td>
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      <td>30.6 +/- 0.8</td>
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  </tr>
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  <tr style="border:1px solid black;">
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      <td>Non-induced overnight culture of KillerRed expressing cells</td>
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      <td>279 +/- 14.5</td>
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      <td>15.5 +/- 0.4</td>
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      <td>31 +/- 0.8</td>
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  </tr>
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  <tr style="border:1px solid black;">
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      <td>Induced growing culture of KillerRed expressing cells</td>
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      <td>8771 +/- 219</td>
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      <td>20.3 +/- 0.4</td>
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      <td>40.6 +/- 0.8</td>
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  </tr>
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  <tr style="border:1px solid black;">
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      <td>Induced overnight culture of KillerRed expressing cells</td>
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      <td>31313 +/- 1075</td>
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      <td>32.5 +/- 0.4</td>
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      <td>65 +/- 0.8</td>
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  </tr>
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</table>
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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>
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<p id="legend">Figure 6.<br>Characterization of the fluorescence measurements</p>
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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.
                                         </p>
                                         </p>
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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 cell, our device needs to control light intensity. To do so, we make this 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>
</p>
</p>
<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>
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                                         <p id="legend"><strong><em>Electronic circuit that enables us to control light intensity</em></strong></br>
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                                         <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>                                       
</p>
</p>
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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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This first part enables us to stabilize the current in the LED lamp. But to control the light intensity, we plug an MOS transistor between the first part and the ground whose gate is plug into a Pulse Width Modulation (PWM) output of Arduino. In this configuration, the transistor work as a switch; it is opened when the gate is at Low level (the ground here) and it is closed when the gate is at a high level (5V here). By modulating the time the circuit is opened per periods we can change the average intensity of the light. The figure below gives 3 examples of this system.</br></br></p>
This first part enables us to stabilize the current in the LED lamp. But to control the light intensity, we plug an MOS transistor between the first part and the ground whose gate is plug into a Pulse Width Modulation (PWM) output of Arduino. In this configuration, the transistor work as a switch; it is opened when the gate is at Low level (the ground here) and it is closed when the gate is at a high level (5V here). By modulating the time the circuit is opened per periods we can change the average intensity of the light. The figure below gives 3 examples of this system.</br></br></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/0/01/PWM_arduino.PNG" alt="PWM_explanation" width="600px" /></p>
<p align="center"><img src="https://static.igem.org/mediawiki/2013/0/01/PWM_arduino.PNG" alt="PWM_explanation" width="600px" /></p>
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                                         <p id="legend"><strong><em>Influence of Pulse Width Modulation(PWM) on the Average Light Intensity</em></strong></br>
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                                         <p id="legend">Figure 8.<br>Influence of Pulse Width Modulation(PWM) on the Average Light Intensity</br>
T: period of the signal; I0: maximum light intensity.</br>
T: period of the signal; I0: maximum light intensity.</br>
The three examples above shows that when the duty cycle of the pulse width modulation changes, the average light intensity changes too. The percentage of the light intensity compared to the maximum I0 is given by the percentage of the duty cycle of the PWM.</br></br>                                       
The three examples above shows that when the duty cycle of the pulse width modulation changes, the average light intensity changes too. The percentage of the light intensity compared to the maximum I0 is given by the percentage of the duty cycle of the PWM.</br></br>                                       
</p>
</p>
<p>
<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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                                     <h2 id="Servo">Servomotor</h2>
                                     <h2 id="Servo">Servomotor</h2>
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                                       <p>The servomotor is used to change the filter in front of the LED Lamp. Arduino actually possesses a library that provides a lot of functions to control the servomotor. We were inspired by <a href="http://www.mon-club-elec.fr/pmwiki_mon_club_elec/pmwiki.php?n=MAIN.ArduinoExpertServoControlePositionClavierPC">this tutorial on a french website</a>.</br>
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                                       <p>The servomotor is used to change the filter in front of the LED Lamp. Arduino actually possesses a library that provides several functions to control the servomotor. We were inspired by <a href="http://www.mon-club-elec.fr/pmwiki_mon_club_elec/pmwiki.php?n=MAIN.ArduinoExpertServoControlePositionClavierPC">this tutorial on a french website</a>.</br>
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After knowing how to control the servomotor, we needed to make some calculation to find the right size of the radius of the servomotor ans the arm that moves the filter rack. Since we are only using 3 different wavelengths, green for the fluorescence, red for inducing KillerRed and white light to kill the cell, we just need 3 slots. To be easier to calculate, we assign the first slot to the first position and the third slot to the last position.</p>
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After discovering how to control the servomotor, we needed to make some calculation to find the right size of the radius of the servomotor and the arm that moves the filter rack. Since we are only using 3 different wavelengths, green for the fluorescence, red for inducing KillerRed, and white light to kill the cell, we only need 3 slots. To make calculations easier, we assign the first slot to the first position and the third slot to the last position.</p>
<p align="center">
<p align="center">
<img src="https://static.igem.org/mediawiki/2013/d/da/Servo_pos1.png" alt="Position1_servo" width="450px" />
<img src="https://static.igem.org/mediawiki/2013/d/da/Servo_pos1.png" alt="Position1_servo" width="450px" />
<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>
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<p id="legend"><strong><em>On the left, the first position of the servomotor and on the right, the second position of the servomotor </em></strong></br>
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<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>
 +
<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>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/d/dd/Box_parts.png" alt="box_parts" width="500px"><p id="legend"><strong><em>Main parts of the device</em></strong></br>
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<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>
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<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 by two excitation filters – green and blue two emission filters – red and yellow and two dichroic mirrors.  We re-design it on SketchUp by adding two more 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 slot 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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<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>
The filters were taken from the cube filter we receive but the plate mirror was created in a clean room by aluminum sputtering  at 70W and 1.2Pa – the thickness of the aluminum is about 20nm.
The filters were taken from the cube filter we receive but the plate mirror was created in a clean room by aluminum sputtering  at 70W and 1.2Pa – the thickness of the aluminum is about 20nm.
</p>
</p>
<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. We need therefore a part that can hold it. We create a mold where you just plug the objective in 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></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 needs to be securely attached on the box. We design a part with two arms where you can screw the servomotor and a flat support to bold it on the box. We drill many holes so that we can easily adjust the height of the servomotor.</p>
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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>
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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>
                                 </li>
                                 </li>

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"