Team:TU-Delft/Zephyr

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Latest revision as of 12:52, 27 October 2013

Zephyr: DIY low-cost fluorescence scanner

Zephyr is a low-cost Do It Yourself (DIY) machine which can scan petridishes and 96 well plates for expression of fluorescence at micrometer scale. The Typhoon is the commercial machine that does the same, only it is priced around 120.000 dollars. The main difference is the use of low-cost optics. This allows you to pick exactly which fluorescence you want to detect and not to pay for the ones you do not use. Furthermore, it does not have confocal optics, as this is not that often when scanning bacteria and protein gels. This DIY machine can be built by anyone with one or two days on their hands and the costs are around 1500 dollars.

The machine is built from a plastic frame, machined by laser-cutting. This is a widely available technique and can be done by many companies. The resulting parts can be assembled like a puzzle, clicking the parts together, making it accessible. The petridishes/gels/plates are moved on a 2D table under an optical tube resembling a fluorescent microscope. By taking images one after another and combining them with the supplied stitching software a high resolution image of the entire object is obtained.

Why? Reason d’être

Research is not cheap and synthetic biology is no exception. Much of the lab equipment has a running price of ten thousand dollars. For some teams this is no hurdle, their lab has all the equipment they possibly may need, while other teams may struggle with their characterization because of lack of needed equipment. This may be an explanation why in the iGEM competition certain regions/continents (e.g. Africa and Latin America) have few teams and this over the past recent years. [1][2] In our view, being able to participate in the iGEM competition should be accessible to everyone and the cost of equipment should not come to hinder creativity all over the world.

For most of the mentioned equipment, only the high tech versions are available, which makes it so costly. However the simple versions of these machines would be enough to carry the work an iGEM team has to do. For instance, we would like to draw a parallel: there are only high tech Bentleys available and no Ford Fiestas, while Fiestas would be enough for simple transportation. This is why we decided to build a low-cost Typhoon, which would be easy to make on your own. This machine is of course not as high-tech as the Typhoon, but it measures at the same scale and has roughly the same performance.

Affordable lab tools for everyone is primordial to making synthetic biology open, accessible, and innovative. As part of our Human Practice endeavor, we wanted to try to make one of these essential tools affordable in order to allow more teams to participate in iGEM in the future. We believe the more teams can participate the more we will all be able to share and build together on new ideas.In the next sections we show the working principle, how to build the Zephyr, the explanation of the design, the results, discussion and conclusion.

What? Working principle

The Zephyr is shown in Figure 2, where the optical parts are in the black tube. In Figure 1 this optical set-up is schematically shown. In this figure the fluorescent object (e.g. cell with GFP) is at the bottom and excited with a LED through an excitation filter. The emitted fluorescence passes through the objective, dichroic mirror, emission filter and eyepiece to be detected using a webcam.
In Figure 2, the excitation is seen as the small blue spot on the 2D table. The 2D table contains a petridish that can be moved around to image the entire plate.
This movement is shown in Figure 3, you move in a snake wise manner around the entire plate to make an image of it. Note that this is not a continuous motion, but a step-wise one. So, the plate is given a small displacement, it is stopped allowing the webcam to take a sharp image and then moved again to take the next image.

Figure 1: Schematic of the optical set-up; Figure 2: Foto of the motion of the Zephyr

Figure 3: Schematic of the scanning motion

How? The Zephyr DIY guide

How to make the Zephyr can be broken down in different modules: first the buying of materials and parts, then the making of several parts, assembling them, wiring the electronic circuit, programming the microprocessor, controlling the set-up from the pc and calibrating the image stitching to make a complete image. The explanation on this is for readability on this separate page.

Explanation of the design

The design of the Zephyr is of course not thoughtless one, many considerations took place. Several of them are listed below, as to explain the design:

As a machining technology the laser cutting is chosen, because it is a technique that is widely available and can be done by a company for a reasonable price (around 150 euros). This way the user does not have to have experience in milling or similar techniques. The same goes for the assembly, by using a click-wise assembly the accessibility of it is high.
The frame material is chosen as PMMA, because it is one of the plastic materials that yields the highest accuracy with laser cutting.
For excitation of the cells a high power LED is chosen over a laser. A laser would give a higher power excitation, but the LED is enough to excite colonies. Since the LED is an order 10 to 100 cheaper it is chosen.
In assembly A, the dichroic holder, next to a dichroic mirror a excitation and emission filter are used. This is common practice in fluorescence microscopy [4], since the selectivity and sensitivity of the measurement go up: the cells are excited with a more precise wavelength and a narrower region of wavelength is measured.
The maximum dimension of the object to be scanned are 140 cm by 140 cm. This is big enough for many protein/DNA gels, petridishes and can fit a 96 well plate.
For actuation stepper motor are chosen as actuation, as they provide relative accurate displacement. The disadvantage is that it uses a lot of power, even when the motor is not moving. Note that 4 motors instead of 2, which would be enough to actuate in two directions. In a first prototype 2 motors were used, however this made the displacement of the 2D table wobbling.
No displacement sensor was used, to improve the accuracy an accelerometer was tested. However this accelerometer in combination with Arduino could not provide high frequency measurements. This had as a result that the measurements did not correlate with the discontinuous displacement.

Results

To test the performance of the Zephyr, three experiments were performed. The first experiment is to test the selectivity: how is an E. coli colony with constitutive GFP expression seen with respect to a colony without GFP and a colony with constitutive RFP expression?

The second experiment is to test the sensitivity: what levels of fluorescence can be detected. This is done with a nucleic acid stain at different concentrations. Finally, a part of a plate with E. coli colonies with constitutive GFP expression is imaged to test the scanning capabilities of the Zephyr.

Selectivity

How selective is the imaging, do you see much background at objects other than GFP? To test this we made a plate as in Figure 12, which is divided into three partitions: one with E. coli with constitutive GFP expression, on E. coli no GFP expression and one with E. coli constitutive RFP expression. The resulting images are also shown in Figure 4 (the black boxes). The GFP picture was unfortunately somewhat out of focus, but the bright shot is the GFP being detected. The two dark pictures have no detection at all.

Figure 4: Image of three partitions: E. coli with constitutive GFP expression, E. coli with constitutive no GFP expression, E. coli with constitutive RFP expression and there the images taken by the Zephyr in the black boxes.

Sensitivity of imaging

To test the sensitivity of the Zephyr, the YOYO1 dye is used. This is a nucleic acid stain that shows fluorescence in the presence of DNA. [3]. This stain shows fluorescence at 510nm, very similar to GFP. This way we use different concentrations of this stain to characterize the sensitivity of the Zephyr to detect fluorescence. The dye is recommended to use at 100 nM. Thus a range of dilutions is made from 2µM to 10nM in water. To all these solutions 500ng of DNA was added. As a control, the 500ng of DNA diluted in water is used.

All these solutions were then scanned by both the Typhoon and the Zephyr, leading to the results of Figure 5. In these bright spots are the fluorescence being detected.

Figure 5: Image of different concentrations of YOYO1 dye, the one on the left being most concentrated. The ‘DNA’ is the control without fluorescent dye. The top lane are the results of the Typhoon and the bottom lane are the results of the Zephyr.

Petridish reading

As explained in the ‘How?’ section, for petridish reading first a calibration must be done. This is done using the calibration text of Figure 6. Using this text (and without the assembly A present), 25 rows of 25 images are scanned. The calibration software finds the displacements between them and first stitches the individual rows together as in Figure 7. Pasting all the individual rows together is done in Figure 8.
Now that the pattern of the displacements is found through this text calibration a part of a plate containing E. coli colonies with constitutive GFP expression, Figure 9, is scanned. The resulting image of this scanning is in Figure 10.

Figure 6: Example of calibration text on the 2D table of the Zephyr, with a 5 eurocent coin as reference.

Figure 7: Row of calibration text stitched together (25 individual pictures)

Figure 8: Rows of calibration text stitched together (25 rows of 25 pictures: 625 pictures)

Figure 9: The part of the plate with E. coli colonies with constitutive GFP expression which is scanned

Figure 10: The resulting image of the scanning of Figure 9.
Video 1: Impression of the Zephyr scanning the calibration text. The Zephyr was displayed on the Discovery Festival to the public, good discussions took place on the mechanism and structure.

Discussion

The selectivity of the Zephyr is good, you only see the expression of GFP, and other fluorescent proteins like RFP do not seem to influence the retrieved image. This is to be expected, since the chosen filters and dichroic mirror are of high quality and are also used in fluorescent microscopy.

The sensitivity of the system is equally good as the Typhoon. It detects the same concentrations and does not detect the same concentrations. Thus the sensitivity of the imaging of the Zephyr is good.

The petridish reading is a difficult task and this becomes clear from the pictures. The text stitched together from 625 individual images is not very successful: the rows do not fully overlap. This is a result of the relative measure: the pictures only have information of their displacement with respect to their neighbor, thus errors will add up over a large amount of pictures. In Figure 10, some colonies are clearly visible, however the alignment is not perfect which is due to the same reason. Adding a sensor to help get a better alignment would greatly improve the performance on this scanning. For more on this, see the section ‘future aspirations’.

Conclusions

The selectivity of the Zephyr is good; it is seeing one type of fluorescence protein at a time. The sensitivity is alright, it can still see a low amount of fluorescence (10 nM YOYO dye). The petridish scanning is working, however the images are not stitched together very well, which results in a fuzzy image.

Future aspirations

Based on these results some improvements can be thought of. The most important one is to improve the petridish reading. This requires a better detection of the displacement. As discussed in the section ‘Explanation of the design choices’, the relative sensing with an accelerometer did not work, probably due to the discontinuous short movements. An alternative to this is using an absolute measurement using a grid. This is schematically shown in Figure 11, where an extra camera is added under the 2D table. This is capturing the grid, so from these images you can find the absolute positions. In this way the image retrieved is more precise.
The image stitching in Matlab works well, only it is relatively slow. This is probably due to the recurrent use of the function nanmean, which is not numerical optimal. Thus an improvement to speed up the image stitching would be to make optimize this function numerically.

Figure 11: Schematic of the improvement of the absolute displacement measurement using a second camera.

References

iGEM.org “Teams Registered for iGEM 2012”,[Online]. Available From: https://igem.org/Team_List?year=2012 viewed on 1 Oct. 2013.
iGEM.org “Teams Registered for iGEM 2013”,[Online]. Available From: https://igem.org/Team_List?year=2013 viewed on 1 Oct. 2013.
Molecular Probes “Dimeric Cyanine Nucleic Acid Stains” at Life Technologies Manuals, Jan-2000
K.R. Spring, "Introduction to Fluorescence Microscopy", [Online]. Available From: http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html viewed on 2 Oct. 2013

@@ Line 18: / Line 18: @@
 <h2 align="center">Why? Reason d’être </h2>
 <p align="justify">
-Research is not cheap in general and synthetic biology is no exception. Much of the lab equipment has a price running of ten thousand dollars. For some teams this is no hurdle, their lab has all the equipment they possibly may need, while other teams may struggle with their characterization because of lack of needed equipment. This may be an explanation why in the iGEM competition certain regions/continents (e.g. Africa and Latin America) have few teams and little very little growth. <a href="https://2013.igem.org/Team:TU-Delft/Zephyr#references" style="text-decoration: none"">[1][2]</a> In our view, being able to participate in the iGEM competition should be accessible to everyone.
+Research is not cheap and synthetic biology is no exception. Much of the lab equipment has a running price of ten thousand dollars. For some teams this is no hurdle, their lab has all the equipment they possibly may need, while other teams may struggle with their characterization because of lack of needed equipment. This may be an explanation why in the iGEM competition certain regions/continents (e.g. Africa and Latin America) have few teams and this over the past recent years. <a href="https://2013.igem.org/Team:TU-Delft/Zephyr#references" style="text-decoration: none"">[1][2]</a> In our view, being able to participate in the iGEM competition should be accessible to everyone and the cost of equipment should not come to hinder creativity all over the world.
 </p>
 <p align="justify">
-For most of the mentioned equipment, only the high tech versions are available, which make it so costly. However the simple versions of these machines would be enough in most cases. As an analog: there are only high tech Bentleys available and no Ford Fiestas, while these Fiestas would be enough for simple transportation.
+For most of the mentioned equipment, only the high tech versions are available, which makes it so costly. However the simple versions of these machines would be enough to carry the work an iGEM team has to do. For instance, we would like to draw a parallel: there are only high tech Bentleys available and no Ford Fiestas, while Fiestas would be enough for simple transportation. This is why we decided to build a low-cost Typhoon, which would be easy to make on your own. This machine is of course not as high-tech as the Typhoon, but it measures at the same scale and has roughly the same performance.
 </p>
 <p align="justify">
-Therefore we decided to build a low-cost Typhoon, which would be easy to make on your own. This machine is of course not as high-tech as the Typhoon, but it measures at the same scale and has roughly the same performance.
+Affordable lab tools for everyone is primordial to making synthetic biology open, accessible, and innovative. As part of our Human Practice endeavor, we wanted to try to make one of these essential tools affordable in order to allow more teams to participate in iGEM in the future. We believe the more teams can participate the more we will all be able to share and build together on new ideas.In the next sections we show the working principle, how to build the Zephyr, the explanation of the design, the results, discussion and conclusion.
 </p>
 <h2 align="center">What? Working principle </h2>
 <p align="justify">
-</p>
+The Zephyr is shown in Figure 2, where the optical parts are in the black tube. In Figure 1 this optical set-up is schematically shown. In this figure the fluorescent object (e.g. cell with GFP) is at the bottom and excited with a LED through an excitation filter. The emitted fluorescence passes through the objective, dichroic mirror, emission filter and eyepiece to be detected using a webcam. <br>
+In Figure 2, the excitation is seen as the small blue spot on the 2D table. The 2D table contains a petridish that can be moved around to image the entire plate. <br>
+This movement is shown in Figure 3, you move in a snake wise manner around the entire plate to make an image of it. Note that this is not a continuous motion, but a step-wise one. So, the plate is given a small displacement, it is stopped allowing the webcam to take a sharp image and then moved again to take the next image.
-<h2 align="center">How? The Zephyr DIY guide</h2>
-<p align="justify">
-How to make the Zephyr can be broken down in different modules: first the buying of materials and parts, then the making of several parts, assembling them, wiring the electronic circuit, programming the microprocessor, controlling the set-up from the pc and calibrating the image stitching to make a complete image.
-</p>
-<h3 align="center">Part list</h3>
-<p align="justify">
-In Table 1 the parts are listed into three categories: optical-, electrical- and mechanical components with a possible online store to buy the components. The plastic PMMA sheets are difficult to acquire online, it usually works the best to contact a local plastic supplier. Most of the mechanical parts can be swapped out for ones with the same dimensions, e.g. the bearings.
-</p>
-<p align="justify">
-Note that in this list only dichroic parts for GFP are listed, for other wavelengths other parts are necessary. The dichroic parts are the excitation- and emission filter and the dichroic mirror itself. For many fluorescent proteins
-<a href="http://www.edmundoptics.com/optics/optical-filters/bandpass-filters/pre-mounted-fluorescence-filter-cubes/3611" style="text-decoration: none"" target="_blank">Edmund Optics</a> has listed a good choice for these. If your fluorescent protein is not on there, the following guidelines may help you: Find out the emission frequency of your protein, pick the frequency of the 25 mm emission filter as close as possible. Pick the dichroic 25.2 x 35.6mm mirror 20 nm lower than this frequency and the 25 mm excitation filter 40 nm lower than the emission filter.
-</p>
-<p align ="justify">
-In addition to these filters and the mirror, you will also need a high power LED. The emission frequency of this LED should be very close to the frequency of the emission filter. Many of these LEDs are available on <a href="http://www.superbrightleds.com/" target="_blank">superbrightleds.com</a>.
-</p>
-<style type="text/css">
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-    font-size: 60%;
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-</style>
-<br>
-<table class="tableizer-table">
-<tr class="tableizer-firstrow"><th>Product name</th><th>Explanation</th><th>Quantity</th><th>Link</th></tr>
- <tr><td><b>Optical components</b></td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
- <tr><td>520nm Bandpass Filter, 36nm Bandpass, OD6 Blocking, 25mm Dia, Stock No. #67-030</td><td>Emission filter</td><td>1</td><td>
-<a href="http://www.edmundoptics.com/optics/optical-filters/bandpass-filters/fluorescence-bandpass-filters/67-030"  target="_blank">link 1</a>
-</td></tr>
- <tr><td>472nm Bandpass Filter, 30nm Bandpass, OD6 Blocking, 25mm Dia, Stock No. #67-027 </td><td>Excitation filter</td><td>1</td><td>
-<a href="http://www.edmundoptics.com/optics/optical-filters/bandpass-filters/fluorescence-bandpass-filters/67-027"  target="_blank">link 2</a>
-</td></tr>
- <tr><td>495nm Dichroic Filter, 25.2 x 35.6mm, Stock No. #67-079 </td><td>Dichroic mirror</td><td>1</td><td>
-<a href="http://www.edmundoptics.com/optics/optical-filters/longpass-edge-filters/fluorescence-dichroic-filters/67-079"  target="_blank">link 3</a>
-</td>
-</tr>
- <tr><td>4X DIN Plan Commercial Grade Objective, Stock No. #67-706 </td><td>Objective</td><td>1</td><td>
-<a href="
-http://www.edmundoptics.com/microscopy/finite-conjugate-objectives/commercial-grade-standard-microscope-objectives/67-706"  target="_blank">link 4</a>
-</td>
-</tr>
- <tr><td>10X DIN Wide Field Microscope Eyepiece, Stock No. #36-130 </td><td>Eyepiece</td><td>1</td><td>
-<a href="
-http://www.edmundoptics.com/microscopy/eyepieces/wide-field-wf-microscope-eyepieces/36-130"  target="_blank">link 5</a>
-</td></tr>
- <tr><td><b>Electronical components</b></td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
- <tr><td>42mm steppermotors 1.8°, 1A, 0.27 Nm, Bestnr.: 198722 - 89</td><td>Stepper motors</td><td>4</td><td>
-<a href="
-http://www.conrad.nl/ce/nl/product/198722/?insert=89&insertNoDeeplink&productname=42mm-servomotor-18-1A-027-Nm"  target="_blank">link 6</a>
-</td>
-</tr>
- <tr><td>Logitech C270 HD Webcam</td><td>Webcam</td><td>1</td><td>
-<a href="
-http://www.amazon.com/Logitech-Widescreen-Calling-Recording-960-000694/dp/B004FHO5Y6"  target="_blank">link 7</a></td></tr>
- <tr><td>Arduino UNO Rev3, Artikelnummer  17458449</td><td>Arduino microprocessor</td><td>1</td><td>
-<a href="
-https://www.sparkfun.com/products/11021"  target="_blank">link 8</a></td></tr>
-</td></tr>
- <tr><td>EasyDriver Stepper Motor Driver</td><td>Drivers to power steppermotors</td><td>2</td><td>
-<a href="
-https://www.sparkfun.com/products/10267"  target="_blank">link 9</a>
-</td>
-</tr>
- <tr><td>DC adapter, 24 V 1.2A</td><td>Adapter to power steppermotors</td><td>1</td><td>
-<a href="
-http://www.conrad.nl/ce/nl/product/512890/VOLTCRAFT-FPPS-24-27W-Stekkervoeding-stekkervoedingsapparaat-schakelvoeding-met-vaste-spanning-24V-1120mA-27-W-at?queryFromSuggest=true"  target="_blank">link 10</a>
-</td></tr>
- <tr><td>XPE series Cree LED blue</td><td>High power LED</td><td>1</td><td>
-<a href="
-http://www.superbrightleds.com/moreinfo/high-powered/xpe-series-cree-led/325/"  target="_blank">link 11</a>
-</td></tr>
- <tr><td>USB-kabel A/B 1.80m</td><td>USB cable to connect with pc</td><td>1</td><td>
-<a href="
-https://www.sparkfun.com/products/512"  target="_blank">link 12</a>
-</td></tr>
- <tr><td>Resistor 5W 5,6Ohm </td><td>Resistor for the LED</td><td>1</td><td>
-<a href="
-http://www.conrad.nl/ce/nl/product/410080/?insert=89&insertNoDeeplink&productname=Vermogensweerstand-56-Axiaal-bedraad-5-W-1-st"  target="_blank">link 13</a>
-</td></tr>
- <tr><td>Rocker Switch </td><td>On / Off switch</td><td>1</td><td>
-<a href="
-https://www.sparkfun.com/products/10727"  target="_blank">link 14</a>
-</td></tr>
- <tr><td>Electrical wire</td><td>Electrical wire to connect parts</td><td>-</td><td></td></tr>
- <tr><td><b>Mechanical parts</b></td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
- <tr><td>PMMA clear 6mm thick, 870 mm x 540 mm</td><td>The plastic for the frame</td><td>1</td><td>-</td></tr>
- <tr><td>PMMA clear 6mm thick, 515 mm x 290 mm</td><td>The plastic for the frame</td><td>1</td><td>-</td></tr>
- <tr><td>PMMA clear 3mm thick, 300 mm x 150 mm </td><td>The plastic for the frame</td><td>1</td><td>-</td></tr>
- <tr><td>Linear bearring 15 mm 8 mm 24 mm</td><td>Bearrings allowing sliding</td><td>8</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/216992/Lineair-kogellager-15-mm-8-mm-24-mm"  target="_blank">link 15</a>
-</td></tr>
- <tr><td>Pouley 30 tooths, 6 mm diameter hole</td><td>Pouley to move the belt</td><td>8</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/209515/?insert=89&insertNoDeeplink&productname=Tandriemschijf-30"  target="_blank">link 16</a>
-</td></tr>
- <tr><td>Belt 950 mm, 380 tooths</td><td>Drive belt</td><td>4</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/209531/?insert=89&insertNoDeeplink&productname=Vlakke-tandriem----950----380"  target="_blank">link 17</a>
-</td></tr>
- <tr><td>Axis 8MM diameter 312 mm length</td><td>C19</td><td>4</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/237205/?insert=89&insertNoDeeplink&productname=Zilverstalen-as"  target="_blank">link 18</a>
-</td></tr>
- <tr><td>Axis 8MM diameter 335 mm length</td><td>C20</td><td>2</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/237205/?insert=89&insertNoDeeplink&productname=Zilverstalen-as"  target="_blank">link 19</a>
-</td></tr>
- <tr><td>Bearring inside diameter 6 mm outer diameter 19 mm </td><td>Bearrings to hold pouleys</td><td>4</td><td>
-<a href="
-http://www.conrad.nl/ce/nl/product/198857/?insert=89&insertNoDeeplink&productname=UBC-Bearing-626-2RS-Radiaalkogellager-600-serie-Boorgatdiameter-6-mm-Buitendiameter-19-mm-Toerental-22000-omwmin"  target="_blank">
-link 20 </a>
-</td></tr>
- <tr><td>Fixation rings 8 mm</td><td>To fixate the axis</td><td>12</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/225550/?insert=89&insertNoDeeplink&productname=Stelringen</td></tr>"  target="_blank">link 21</td></tr></a>
- <tr><td>SM1V10 - Ø1" SM1 Lens Tube, 1" Long External Threads</td><td>Lens tube allowing focussing</td><td>1</td><td>
-<a href="http://www.thorlabs.de/newgrouppage9.cfm?objectgroup_id=4109&pn=SM1V10#3389</td></tr>"  target="_blank">link 22</td></tr></a>
-</td></tr>
- <tr><td>CP4S - SM1-Threaded 30 mm Cage Plate, 4 mm Thick</td><td>Plate mounting for the lens</td><td>1</td><td>
-<a href="http://www.thorlabs.de/newgrouppage9.cfm?objectgroup_id=2273&pn=CP4S#2761</td></tr>"  target="_blank">link 23</td></tr></a>
-</td></tr>
- <tr><td>SM1A3 - Adapter with External SM1 Threads and Internal RMS Threads</td><td>Adapter between different threads </td><td>1</td><td>
-<a href="http://www.thorlabs.de/newgrouppage9.cfm?objectgroup_id=1524&pn=SM1A3#5081</td></tr>"  target="_blank">link 24</td></tr></a>
-</td></tr>
- <tr><td>M6 bolts x 40 mm +nuts</td><td>4 to support the pouleys and 4 for the holding of the objectie plate</td><td>8</td><td>-</td></tr>
- <tr><td>M3 bolts x 20 mm + nuts</td><td>To tightent belts to sliders</td><td>8</td><td>-</td></tr>
- <tr><td>Black paint</td><td>e.g. exhaust paint</td><td>1</td><td>-</td></tr>
- <tr><td>Heat transfer double sided tape</td><td>To fixate the high power LED</td><td>1</td><td>
-<a href="http://www.conrad.nl/ce/nl/product/358384/?insert=89&insertNoDeeplink&productname=Warmtegeleidende-kleeffolie-TCT-SEPA-TCT35-1-WmK-Dikte-025-mm</td></tr>"  target="_blank">
-link 25</td></tr></a>
- <tr><td>M3 bolts x 15 mm</td><td>To mount motors</td><td>16</td><td></td></tr>
- <tr><td></td><td></td><td>1</td><td>-</td></tr>
- <tr><td></td><td>1</td><td></td></tr></a>
-</td></tr>
- <tr><td>Glue</td><td>To fixate webcam</td><td>-</td><td></td></tr>
-</table>
 <center>
-<p>
+	<img src="https://static.igem.org/mediawiki/2013/c/c6/Figure_1_optical_set_up.jpg"/ height="350px">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-Table 1: The parts to buy of the Zephyr, including dichroic parts for GFP detection.
+	<img src="https://static.igem.org/mediawiki/2013/d/d3/2D_table_Zephyr.jpg"/ height="350px">
-</p>
 </center>
+<center>Figure 1: Schematic of the optical set-up;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure 2: Foto of the motion of the Zephyr</center>
-<h3 align="center">Making of the parts</h3>
-<p align="justify">
-In total 41 unique parts must be made out of plastic using laser cutting.
-These parts are dived into four categories: A to D. A are the parts of the dichroic holder including the LED holder. B are the parts of the optical holder, C are the frame parts and D are the parts that hold petridishes and the 96 well plates. In the table below the parts are listed with their name and their coding (e.g. B3).
-</p>
-<br>
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/b/be/JoinedDelft.jpg" width="850px" height="772px"/>
+	<img src="https://static.igem.org/mediawiki/2013/4/4d/Figure_3_movement.jpg"/ height="350px">
-</center>
-<center>Table 2: The parts to make for the Zephyr. </center>
-<p align="justify">
-For all these parts technical drawings are available below or bundled in this<a href="https://static.igem.org/mediawiki/2013/2/2f/Zephyr_part_drawings.pdf"  target="_blank"> pdf</a>. Note that these are the dimensions that result from using the laser cutting method.
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-<script src="http://thecodeplayer.com/uploads/js/prefixfree.js" type="text/javascript"></script>
-<br>
-Figure 4: The Individual technical drawings of the parts to make
-<br>
-<br>
-So, how to make these parts? For laser cutting the parts to make must usually be supplied a ‘dxf’-format, this is a file containing the 2D structure of the different parts. For all the different parts these files can be found in this
-<a href="https://2013.igem.org/File:Individual_dxfs.zip"  target="_blank"> zip-file</a>. These digital files can be directly sent to a company that can make them for you or a technician at a university. The three plastic plates will suffice to make all the parts according to the quantity. You will have to ask them to combine them in a smart way for you on the plate. This would look something in Figure 5.
-<br>
-<center>
-<img src="https://static.igem.org/mediawiki/2013/0/02/Figure_2_plate_C.png" />
-<br>
-Figure 5: Example of the collection of the different parts in the laser program
-<br><br>
-<img src="https://static.igem.org/mediawiki/2013/b/bb/Figure_3_laser_result.jpg" />
-<br>
-Figure 6: Example of the parts being lasered out a PMMA plate
-<br>
 </center>
+<center>Figure 3: Schematic of the scanning motion</center>
 </p>
+<h2 align="center">How? The Zephyr DIY guide</h2>
 <p align="justify">
-After these parts are cut, all the A and B parts must be painted. Paint both of the sides like in Figure 7, this will prevent reflection of light inside the optical tube and interference of outside light.
+How to make the Zephyr can be broken down in different modules: first the buying of materials and parts, then the making of several parts, assembling them, wiring the electronic circuit, programming the microprocessor, controlling the set-up from the pc and calibrating the image stitching to make a complete image. The explanation on this is for readability on <a href="https://2013.igem.org/Team:TU-Delft/Zephyr_How">this</a> separate page.
-<br>
-<center>
-<img src="https://static.igem.org/mediawiki/2013/b/bc/Figure_4_painted_parts.png" />
-<br>
-Figure 7: Example of painting the A- parts.
-<br>
-</center>
-</p>
-<h3 align="center">Assembly of the parts</h3>
-<br>
-<p align="justify">
-The assembly is described in the images below in a step-wise manner. Before starting, the webcam must be modified and the LED wired, see the file on preparation. After this it is sequentially assembling A, B and C.
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-<div class="img" align="center">
- <a target="_blank" href="https://static.igem.org/mediawiki/2013/4/40/Building_1.jpg"><img src="https://static.igem.org/mediawiki/2013/4/40/Building_1.jpg" alt="Assembly of A" width="300" height="420"></a>
- <div class="desc">Assembly of A</div>
-</div>
-<div class="img">
- <a target="_blank" href="https://static.igem.org/mediawiki/2013/1/1f/Building_2.jpg"><img src="https://static.igem.org/mediawiki/2013/1/1f/Building_2.jpg" alt="id1" width="300" height="420"></a>
- <div class="desc">Assembly of B</div>
-</div>
-<div class="img">
- <a target="_blank" href="https://static.igem.org/mediawiki/2013/8/84/Preparation.jpg"><img src="https://static.igem.org/mediawiki/2013/8/84/Preparation.jpg" alt="id2" width="300" height="420"></a>
- <div class="desc">Preparation of webcam and LED</div>
-</div>
-</div>
-<p align="justify">
-<center>
-Figure 8: Step-wise explanation of the assembly.
-</center>
-</p>
-<br>
-<h3 align="center">Wiring the circuits</h3>
-<p align="justify">
-Now that everything is assembled, the electrical circuit must be made. It consists of 4 major components: the Arduino, the motor drivers, the motors and the LED. The LED is connected in series with a resistor to the Arduino, and the motors via the drivers to the Arduino. The total schematic is shown in Figure 9. Note that the motors in one direction are connected in series, since the motion must be the same for these two motors. For example if you want to move the table along the x-axis two motors should move: one pulling the drive belt and one pushing the drive belt. The wiring colors of stepper motors are not uniform, so you will have to do some trial and error to get this working correctly.
-</p>
-<h3 align="center">Software</h3>
-<p align="justify">
-The Arduino controls both the LED, all the motors and communicates with the PC. Attached is the zip [linkto:software_Zephyr] file containing all the software, including the Arduino code file. This file can be uploaded to the Arduino using the Arduino <a href= “http://arduino.cc/en/Main/Software”>software</a>. It starts the scanning at the command of the PC and sends the time instances the photo must be taken.
-On the pc, the control software is written in <a href= “http://www.microsoft.com/visualstudio/eng”>Visual C++</a>. Upon starting it opens a command window in which the scanning can be started and through which the webcam images are saved in a folder on the hard drive.
-</p>
-<p align="justify">
-The image processing is done in Matlab in two steps. Our experience shows that the displacements between the pictures are unfortunately not constant. To deal with this we designed stitching software that finds the ROI of pictures and the displacements between pictures and then pastes them together, see for an example Figure 10. The two steps are first a calibration using a text and then the actual scanning using the fluorescent filters. This calibration text is a text with a small font (e.g. 2pt) which allows the stitching program to have enough features to find the correct displacements between the pictures. This scanning is done without the dichroic module (subassembly A) present. An example of this text is in Figure 11, a 5 eurocent coin is added for reference. Once the pattern of displacements is found, the Zephyr can scan the petridish on fluorescence and stitch the images together using this found pattern.
-</p>
-<center>
-	<img src="https://static.igem.org/mediawiki/2013/a/ab/Figure10_example_stitching.png"/>
-</center>
-<center>Figure 10: Example of finding the displacements between two pictures and overlapping them. </center>
-<center>
-	<img src="https://static.igem.org/mediawiki/2013/5/51/Figure11_expampletext.JPG"/ width="600px" height="450px">
-</center>
-<center>Figure 11: Example of calibration text on the 2D table of the Zephyr, with a 5 eurocent coin as reference.</center>
 <h2 align="center">Explanation of the design </h2>
 <p>
+The design of the Zephyr is of course not thoughtless one, many considerations took place. Several of them are listed below, as to explain the design:
 <ul>
-<li>	Use of laser cutting</li>
+<li>As a machining technology the laser cutting is chosen, because it is a technique that is widely available and can be done by a company for a reasonable price (around 150 euros). This way the user does not have to have experience in milling or similar techniques. The same goes for the assembly, by using a click-wise assembly the accessibility of it is high. </li>
-<li>	Clicking the parts together -> accessible, fun, enough stiffness</li>
+<li>The frame material is chosen as PMMA, because it is one of the plastic materials that yields the highest accuracy with laser cutting. </li>
-<li>	Stepper motor -> accurate displacement, however high power use</li>
+<li>For excitation of the cells a high power LED is chosen over a laser. A laser would give a higher power excitation, but the LED is enough to excite colonies. Since the LED is an order 10 to 100 cheaper it is chosen. </li>
-<li>	High power LED -> cheaper than laser</li>
+<li>In assembly A, the dichroic holder, next to a dichroic mirror a excitation and emission filter are used. This is common practice in fluorescence microscopy [4], since the selectivity and sensitivity of the measurement go up: the cells are excited with a more precise wavelength and a narrower region of wavelength is measured. </li>
-<li>	Motivation size -> what can fit</li>
+<li>The maximum dimension of the object to be scanned are 140 cm by 140 cm. This is big enough for many protein/DNA gels, petridishes and can fit a 96 well plate.</li>
-<li>	Use of filters next to the dichroic mirror -> high selectivity/sensitivity</li>
+<li>For actuation stepper motor are chosen as actuation, as they provide relative accurate displacement. The disadvantage is that it uses a lot of power, even when the motor is not moving. Note that 4 motors instead of 2, which would be enough to actuate in two directions. In a first prototype 2 motors were used, however this made the displacement of the 2D table wobbling.</li>
-<li>	Use of 4 motors instead of 2 -> woggly movement</li>
+<li>No displacement sensor was used, to improve the accuracy an accelerometer was tested. However this accelerometer in combination with Arduino could not provide high frequency measurements. This had as a result that the measurements did not correlate with the discontinuous displacement.</li>
-<li>	Fitting of the axis -> imperfection of laser cutting</li>
+</ul>
-<ul>
 </p>
 <h2 align="center">Results</h2>
 <p>
-To test the performance of the Zephyr, three experiments were performed. The first experiment is to test the selectivity: how is an E.coli colony with constitutive GFP expression seen with respect to a colony without GFP and a colony with constitutive RFP expression?
+To test the performance of the Zephyr, three experiments were performed. The first experiment is to test the selectivity: how is an <i>E. coli</i> colony with constitutive GFP expression seen with respect to a colony without GFP and a colony with constitutive RFP expression?
 </p>
 <p align="justify">
 The second experiment is to test the sensitivity: what levels of fluorescence can be detected. This is done with a nucleic acid stain at different concentrations.
-Finally, a part of a plate with E.coli colonies with constitutive GFP expression is imaged to test the scanning capabilities of the Zephyr
+Finally, a part of a plate with <i>E. coli</i> colonies with constitutive GFP expression is imaged to test the scanning capabilities of the Zephyr.
 </p>
 <h3 align="center">Selectivity</h3>
 <p align="justify">
-How selective is the imaging, do you see much background at objects other than GFP? To test this we made a plate as in Figure 12, which is divided into three partitions: one with E.coli with constitutive GFP expression, one plain BL21 (no GFP expression) and one with E.coli constitutive RFP expression.
+How selective is the imaging, do you see much background at objects other than GFP? To test this we made a plate as in Figure 12, which is divided into three partitions: one with <i>E. coli</i> with constitutive GFP expression, on <i>E. coli</i> no GFP expression and one with <i>E. coli</i> constitutive RFP expression.
-The resulting images are also shown in Figure 12 (the black boxes). The GFP picture was unfortunately somewhat out of focus, but the bright shot is the GFP being detected. The two dark pictures have no detection at all.
+The resulting images are also shown in Figure 4 (the black boxes). The GFP picture was unfortunately somewhat out of focus, but the bright shot is the GFP being detected. The two dark pictures have no detection at all.
 </p>
 <center>
 	<img src="https://static.igem.org/mediawiki/2013/8/8a/Figure12_results_selectivity.jpg"/ width="600px" height="450px">
 </center>
-<center>Figure 12: Image of three partitions: E.coli with constitutive GFP expression, E.coli with constitutive no GFP expression, E.coli with constitutive RFP expression and there the images taken by the Zephyr in the black boxes. </center>
+<center>Figure 4: Image of three partitions: <i>E. coli</i> with constitutive GFP expression, <i>E. coli</i> with constitutive no GFP expression, <i>E. coli</i> with constitutive RFP expression and there the images taken by the Zephyr in the black boxes. </center>
 <br>
-<h3 align="center">Sensitivity</h3>
+<h3 align="center">Sensitivity of imaging</h3>
 <p align="justify">
-To test the sensitivity of the Zephyr, the YOYO1 dye is used. This is a nucleic acid stain that shows fluorescence in the presence of DNA. <a href="https://2013.igem.org/Team:TU-Delft/Zephyr#references" style="text-decoration: none"">[3]</a> This stain shows fluorescence at 510nm, very similar to GFP. This way we use different concentrations of this stain to characterize the sensitivity of the Zephyr to detect fluorescence. The dye is recommended to use at 100 nM, which is a dilution of 10,000x from the stock at 1mM. Thus a range of dilutions of this stock is made from 500x (2µM) to 100,000x (10nM) in water. To all these solutions 500ng of DNA was added. As a control, the 500ng of DNA diluted in water is used.
+To test the sensitivity of the Zephyr, the YOYO1 dye is used. This is a nucleic acid stain that shows fluorescence in the presence of DNA. <a href="https://2013.igem.org/Team:TU-Delft/Zephyr#references" style="text-decoration: none"">[3]</a>. This stain shows fluorescence at 510nm, very similar to GFP. This way we use different concentrations of this stain to characterize the sensitivity of the Zephyr to detect fluorescence. The dye is recommended to use at 100 nM. Thus a range of dilutions  is made from 2µM to 10nM in water. To all these solutions 500ng of DNA was added. As a control, the 500ng of DNA diluted in water is used.
 </p>
 <p align="justify">
-All these solutions were then scanned by the Zephyr, leading to the results of Figure 13. In these bright spots are the fluorescence being detected.
+All these solutions were then scanned by both the Typhoon and the Zephyr, leading to the results of Figure 5. In these bright spots are the fluorescence being detected.
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/9/96/Figure12_yoyo.jpg"/ width="700px" height="105px">
+	<img src="https://static.igem.org/mediawiki/2013/9/96/Figure12_yoyo.jpg"/ width="700px">
 </center>
-<center>Figure 13: Image of different concentrations of YOYO1 dye, the one on the left being most concentrated. The ‘DNA’ is the control without fluorescent dye. </center>
+<center>Figure 5: Image of different concentrations of YOYO1 dye, the one on the left being most concentrated. The ‘DNA’ is the control without fluorescent dye. The top lane are the results of the Typhoon and the bottom lane are the results of the Zephyr. </center>
 <br>
 </p>
-[ref3]: Molecular Probes “Dimeric Cyanine Nucleic Acid Stains” at Life Technologies Manuals, Jan-2000
 <h3 align="center">Petridish reading</h3>
 <p>
-As explained in the <br>
+As explained in the ‘How?’ section, for petridish reading first a calibration must be done. This is done using the calibration text of Figure 6. Using this text (and without the assembly A present), 25 rows of 25 images are scanned. The calibration software finds the displacements between them and first stitches the individual rows together as in Figure 7. Pasting all the individual rows together is done in Figure 8. <br>
+Now that the pattern of the displacements is found through this text calibration a part of a plate containing <i>E. coli</i> colonies with constitutive GFP expression, Figure 9, is scanned. The resulting image of this scanning is in Figure 10. </p>
+<br>
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/6/60/Figure14_row_stitch.jpg"/ width="700px" height="193px">
+	<img src="https://static.igem.org/mediawiki/2013/5/51/Figure11_expampletext.JPG"/ width="500px" >
 </center>
-<center>Figure 14: Row of calibration text stitched together (25 individual pictures)</center>
+<center>Figure 6: Example of calibration text on the 2D table of the Zephyr, with a 5 eurocent coin as reference.</center>
+<center>
+	<img src="https://static.igem.org/mediawiki/2013/6/60/Figure14_row_stitch.jpg"/ width="700px">
+</center>
+<center>Figure 7: Row of calibration text stitched together (25 individual pictures)</center>
 <br>
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/1/19/Figure15_text_stitch.jpg"/ width="700px" height="374px">
+	<img src="https://static.igem.org/mediawiki/2013/1/19/Figure15_text_stitch.jpg"/ width="600px">
 </center>
-<center>Figure 15: Rows of calibration text stitched together (25 rows of 25 pictures: 625 pictures)</center>
+<center>Figure 8: Rows of calibration text stitched together (25 rows of 25 pictures: 625 pictures)</center>
 <br>
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/f/fd/Figure16_Scanning_test.JPG"/ width="700px" height="525px">
+	<img src="https://static.igem.org/mediawiki/2013/f/fd/Figure16_Scanning_test.JPG"/ width="600px">
 </center>
-<center>Figure 16: </center>
+<center>Figure 9: The part of the plate with <i>E. coli</i> colonies with constitutive GFP expression which is scanned</center>
 <br>
 <center>
-	<img src="https://static.igem.org/mediawiki/2013/4/42/Figure17_gfp_plate_stitched.jpg"/ width="700px" height="409px">
+	<img src="https://static.igem.org/mediawiki/2013/4/42/Figure17_gfp_plate_stitched.jpg"/ width="600px">
 </center>
-<center>Figure 17: </center>
+<center>Figure 10: The resulting image of the scanning of Figure 9. </center>
 <br>
+<center><iframe src="https://www.facebook.com/video/embed?video_id=543051369093630" width="600" height="337" frameborder="0"></iframe></center>
+<center>Video 1: Impression of the Zephyr scanning the calibration text.</center>
-</p>
+The Zephyr was displayed on the <a href="https://2013.igem.org/Team:TU-Delft/Summer_Festival" tager="blank">Discovery Festival</a> to the public, good discussions took place on the mechanism and structure.
 <h2 align="center">Discussion</h2>
 <p>
-<ul>
+The selectivity of the Zephyr is good, you only see the expression of GFP, and other fluorescent proteins like RFP do not seem to influence the retrieved image. This is to be expected, since the chosen filters and dichroic mirror are of high quality and are also used in fluorescent microscopy. <br><br>
-<li>	Selectivity:</li>
+The sensitivity of the system is equally good as the Typhoon. It detects the same concentrations and does not detect the same concentrations. Thus the sensitivity of the imaging of the Zephyr is good.<br><br>
-<li>	Sensitivity:</li>
+The petridish reading is a difficult task and this becomes clear from the pictures. The text stitched together from 625 individual images is not very successful: the rows do not fully overlap. This is a result of the relative measure: the pictures only have information of their displacement with respect to their neighbor, thus errors will add up over a large amount of pictures. In Figure 10, some colonies are clearly visible, however the alignment is not perfect which is due to the same reason. Adding a sensor to help get a better alignment would greatly improve the performance on this scanning. For more on this, see the section ‘future aspirations’.
-<li>	Petridish reading:</li>
-<li>	Overall:</li>
-</ul>
 </p>
 <h2 align="center">Conclusions</h2>
 <p>
-<ul>
+The selectivity of the Zephyr is good; it is seeing one type of fluorescence protein at a time. The sensitivity is alright, it can still see a low amount of fluorescence (10 nM YOYO dye). The petridish scanning is working, however the images are not stitched together very well, which results in a fuzzy image.
-<li>	Selectivity:</li>
-<li>	Sensitivity:</li>
-<li>	Petridish reading:</li>
-<li>	Overall:</li>
-</ul>
 </p>
 <h2 align="center">Future aspirations</h2>
 <p>
-<ul>
+Based on these results some improvements can be thought of. The most important one is to improve the petridish reading. This requires a better detection of the displacement. As discussed in the section ‘Explanation of the design choices’, the relative sensing with an accelerometer did not work, probably due to the discontinuous short movements. An alternative to this is using an absolute measurement using a grid. This is schematically shown in Figure 11, where an extra camera is added under the 2D table. This is capturing the grid, so from these images you can find the absolute positions. In this way the image retrieved is more precise. <br>
-<li>	Better Sensor -> higher accuracy.</li>
+The image stitching in Matlab works well, only it is relatively slow. This is probably due to the recurrent use of the function <b>nanmean</b>, which is not numerical optimal. Thus an improvement to speed up the image stitching would be to make optimize this function numerically.
-<li>	Faster reading -> flashing.</li>
+<center>
-<li>	Faster image stitching -> C. </li>
+	<img src="https://static.igem.org/mediawiki/2013/a/a3/Figure11_improvement.jpg"/ width="450px">
-</ul>
+</center>
+<center>Figure 11: Schematic of the improvement of the absolute displacement measurement using a second camera.</center>
+<br>
 </p>
@@ Line 861: / Line 171: @@
   https://igem.org/Team_List?year=2013" style="text-decoration: none"" target="_blank">
   https://igem.org/Team_List?year=2013</a>  viewed on 1 Oct. 2013.
+<li>Molecular Probes “Dimeric Cyanine Nucleic Acid Stains” at Life Technologies Manuals, Jan-2000</li>
+<li>K.R. Spring, "Introduction to Fluorescence Microscopy", [Online]. Available From: <a href="http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html" style="text-decoration: none"" target="_blank">http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html</a> viewed on 2 Oct. 2013
 </li>
-<li>Molecular Probes “Dimeric Cyanine Nucleic Acid Stains” at Life Technologies Manuals, Jan-2000</li>
 </ol>
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