Team:Aachen/OD/F device

From 2014.igem.org

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== Development ==
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== Measuring Principle ==
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Building the OD/F device has been an interesting task. On the one hand, this device has been developed mainly by the IT division of our team. On the other hand, we got assistance from biologists suffering from color-blindness, yet eager to help selecting the best color filters for the LEDs. For the next year, you really have to select carefully who's going to help with which task!
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The measuring principle for both optical density (OD) and fluorescence  measurement is depicted below.
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For OD measurement we shine through the sample with an LED and a fixed width. A filter blocks any other light but 600 nm. This way, the sensor mainly senses the 600 nm light which is needed for OD600 measurement.
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=== Cuvette Holder ===
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For fluorescence measurement a similar approach is chosen. The filter again is used to block the exciting light from being sensed. That way only the emitted light from the fluorescence protein is measured.
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The essential part of this device is the '''cuvette holder which has also been the most tricky thing to design'''. In short, we had to overcome a dilemma created by the need for an optimal height for the sensor:
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* A too low sensor position bears problems with sedimentation as well as light diffraction from the bottom of the cuvette.
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* The sensor has to be as close as possible to the bottom so that enough light shines through for the fluorescence measurement.
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As a compromise, we place the sensor at a height of 0.75 cm, which, as it turned out later, is very close to one of the standard heights (0.2 cm, 0.8 cm, 1.2 cm) of OD meters. It is important to note that despite the official minimal fill height of 1.2 mL of the 1.5 mL cuvettes we used, our device also works with filling volumens of just 1 mL which in fact comes closer to reality in the lab.
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<center>
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{{Team:Aachen/Figure|Aachen_odf_schemes.png|title=Measuring principle for OD/F device|subtitle=The left image shows the measurement approach for the optical density. The light shines through the sample with a fixed width. The right image shows the fluorescence measurement approach, exciting the fluorescence proteins from below and measuring from the side.|width=500px}}
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</center>
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The final cuvette holder design was rendered in a stl-file shown below:
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Further details about selecting filters, code, a construction manual and evaluation can be found [https://2014.igem.org/Team:Aachen/Notebook/Engineering/ODF here].
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<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render
<iframe src="https://2014.igem.org/Team:Aachen/Notebook/Engineering/Cuvette3D?action=render
" width=500px height=500px frameBorder="0"></iframe>
" width=500px height=500px frameBorder="0"></iframe>
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<p>Cuvette Holder for optical density and fluorescence measurement.</p>
</center>
</center>
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=== Light filters ===
 
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Once the cuvette holder was finished, '''finding good filters was a tough challenge'''. A main goal throughout our project has been to choose easily available parts which are also inexpensive. Thus choosing Schott glasses as filters unfortunately could not be considered. Instead, filters used for illumination of theaters seemed to be ideal solution.
 
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Especially for the fluorescence measurements of GFP finding the right filter has been a big problem. [http://parts.igem.org/Part:BBa_E0040 GFPmut3b] has a peak excitation at 501&nbsp;nm and a peak emission at 511&nbsp;nm - too close together for our low-cost filters to block the excitation light but transmit the emitted light. Thus, we chose to excite at around 485&nbsp;nm reduce false positive results below 500&nbsp;nm. However, no adequate filter for these settings could be found.
 
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Eventually, using the dark greenish Twickenham Green filter only little amounts of light shorter than 500&nbsp;nm got through, reducing any bias from excitation illumination significantly. Unfortunately, the transmission rate of this filter is quite bad, 20&nbsp;% only, for the target emission wavelength of 511&nbsp;nm.
 
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For the OD measurement, too, we had similar problems. The solution to this problem is presented in the F device section.
 
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<html>
 
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<sup><span class="anchor" id="fn1"></span>1. Quite a good random number generator from a computer-scientific perspective!<a href="#ref1" title="">↩</a></sup>
 
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<center>
 
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{{Team:Aachen/FigureFlex|Aachen_odf_schemes.png|title=Setup of the measuring unit|subtitle=On the left, the setup of a classical spectrophotometer is depicted. The setup around the cuvette holder of our device is shown on the right.|width=400px}}
 
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== Combined Device ==
== Combined Device ==

Revision as of 09:14, 14 October 2014

OD/F device

Measuring Optical Density (OD) is a central element in microbiological work and synthetic biology. One question that has to be answered often is how many cells are in a suspension. Here, the OD can give you a hint. Unfortunately, commercially available OD meters cost several hundred dollars ([http://www.laboratory-equipment.com/laboratory-equipment/cell-density-meter.php OD meter]), and can limit the spread of synthetic biology.

Therefore, we wanted to devenlop an alternative for measuring OD, specifically designed for Biohackspaces, DIY and community laboratories and schools. With our OD/F device, we want to enable many people to do good, precise and inexpensive science research.

Especially for the Interlab Study fluorescence, too, has been of importance. One aim of this study was to measure the correlation between OD and fluorescence. Since the taks of measuring OD and fluorescence are often performed at the same time, we want to present a device that can measure both fluorescence and OD with just some easy adjustments. This way, we can measure how much fluorescence there is per amount of cells.

In fact, you can find some DIY posts for turbidity meters such as [http://www.thingiverse.com/thing:74415 turbidity sensors]. However, a proper assessment of their linearity as well as a calculated OD-value are missing.

Regarding fluorescence, we are of course not re-inventing the wheel (well, not totally). The 2010 iGEM Cambridge team actually built a very similar device, the E.glometer. However, there's no data available showing an actual comparison of the data from their device and some proven commercial system to, for example, assess linearity of the measurement.

Measuring Principle

The measuring principle for both optical density (OD) and fluorescence measurement is depicted below. For OD measurement we shine through the sample with an LED and a fixed width. A filter blocks any other light but 600 nm. This way, the sensor mainly senses the 600 nm light which is needed for OD600 measurement.

For fluorescence measurement a similar approach is chosen. The filter again is used to block the exciting light from being sensed. That way only the emitted light from the fluorescence protein is measured.

Aachen odf schemes.png
Measuring principle for OD/F device
The left image shows the measurement approach for the optical density. The light shines through the sample with a fixed width. The right image shows the fluorescence measurement approach, exciting the fluorescence proteins from below and measuring from the side.

Further details about selecting filters, code, a construction manual and evaluation can be found here.

Cuvette Holder for optical density and fluorescence measurement.

Combined Device

Even though evaluation of the measurements have been performed in two separate device, it is fairly well possible to put everything into one casing. All you need to do is choosing another lid, and connect a second light to frequency sensor to your Arduino. Right at the bottom we present you the differences in wiring things up.

OD device

Aachen 14-10-09 Flowsheet OD-device ipo.png
How to use our OD/F device
XXX

Contact Disclaimer

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