TITLE: Building a phenotype-o-mat: A low-cost DIY plate reader for high-throughput phenotype measurements
AUTHORS: Ben Braverman

[DESCRIPTION]

 

We've developed a DIY, low-cost, 96-well plate imager called the phenotype-o-mat. This protocol outlines the steps to assemble it yourself. We cover:
1) 3D-printing components
2) Adding threaded inserts 
3) Soldering LEDs
4) Setting up the Arduino Nano Every
5) Connecting the BlackFly camera
6) Running an assay

While you can 3D-print a few of the components, the rest are commercially available. Check out the "Materials" tab to see what you'll need before beginning assembly. The necessary 3D-printing files and code are available in our phenotype-o-mat GitHub repo.

If you have any questions or suggestions about assembly, please feel free to reach out at ben.braverman@arcadiascience.com or comment on our forthcoming phenotype-o-mat pub at research.arcadiascience.com.

[STEPS]
SECTION: 3D-printing components
2. The phenotype-o-mat has the following 3D-printed components:
Plate-holder base
Camera arm
Camera mount
Circular filter slider
LED ring and diffuser mount clip
LED diffuser ring
Arduino Nano Every case (credit: leondl on Thingiverse)

Access STL files for these parts >

 

SECTION: 3D-printing components
2.1. Plate-holder base

Place the model flat on the print bed and manually place supports under the LED slot. Print with PLA filament using a 15% grid infill.

 

SECTION: 3D-printing components
2.2. Other attachments
You can print all other phenotype-o-mat attachments except the diffuser in black PLA using a 20% grid infill and auto-generated supports. You must print the diffuser using a clear filament.

 

SECTION: Adding threaded inserts
3. The heat-set threaded inserts allow you to add threaded metal screws to both the plate-holder base and the camera arm.

 

 

Place the 8 mm M3 threaded insert into the side hole on the plate-holder base, perpendicular to the surface. Heat a soldering iron to 200 °C and place it on the threaded insert. As the insert heats up, apply light pressure. Allow the insert to melt the plastic around it and push it into place. Make sure not to press the threaded insert too far, as it will obstruct the aluminum extrusion.

Add threaded inserts to the camera arm in the same way. Add 8 mm M3 inserts at the neck attachment point and 4 mm M3 brass inserts at the camera holder.

 

Once you've added the threaded insert, you can add an M3 thumb screw to fasten it to the aluminum extrusion.

SECTION: Soldering LEDs
4. The phenotype-o-mat uses transillumination LED panels to illuminate plates from below and a custom LED ring to illuminate plates with blue, green, yellow, and red LEDs. This section covers how to assemble the LED ring and LED panels. You'll need the following materials:
Soldering iron
Soldering iron helping hands
Solder wire
Three white LED backlight modules (Large, 45 mm × 86 mm)
Five super bright blue 5 mm LEDs
Five super bright red 5 mm LEDs
Five super bright yellow 5 mm LEDs
Five super bright green 5 mm LEDs
18 100-ohm resistors (used for green, blue, and yellow LEDs)
Five 150-ohm resistors (used for red LEDs)

For an introduction to soldering resistors to LEDs, check out this video:
 
Credit: Random Making Encounters

SECTION: Soldering LEDs
4.1. Soldering resistors to LEDs

 

The green, blue, and yellow LEDs need 100-ohm resistors and the red LEDs need 150-ohm resistors.

Snip the longer wire off the LED at the notch near the bulb.
Cut one side of the resistor wire so it is about the same length as the nub of wire left on the LED.
Using grippers, align the short end of the resistor with the short wire on the LED.
Carefully solder these two together.
Set the LED/resistor combo aside, making sure to keep note of the LED’s color.
Test the LEDs with a breadboard to make sure they're working.

 

 

SECTION: Soldering LEDs
4.2. Soldering LEDs with resistors to the PCB ring

After all of the LEDs have a resistor, begin soldering them to the custom-printed circuit board (PCB ring) in the following positions:
 

 

Hold the PCB ring in place with grippers.
Place the LED into the appropriate PCB holes with the resistor side going through the circular hole and the other side going through the square hole. Make sure the PCB is oriented with the labels facing up. Get the resistor as close to the board as possible and try to make all the LEDs about the same height.
Solder these wires to the PCB by carefully heating the metal pads next to the wire, being mindful not to melt the board or accidentally solder the wires together.
Snip the excess wire off and continue soldering until all LEDs are attached.

 

SECTION: Soldering LEDs
4.3. Soldering wires to the PCB

Once the LEDs with resistors are all attached to the PCB ring, wire the entire PCB to the Arduino Nano Every. Get two pieces of black wire and one piece each of white, green, blue, yellow, and red wire. Each piece needs to be about a third of a meter long.

Remove about 2.5 cm of insulation from the ends of each wire using wire strippers.
On the PCB, place the wires in the following positions and solder them carefully to the board. The arrow printed on the left indicates the "ground" position. Wrap the bundle of wires with heat shrink to keep them in place.

 


 


SECTION: Soldering LEDs
4.4. Soldering transillumination LED panels

Each LED panel requires a 100-ohm resistor. You can solder them to the wires of the LED panel in the same way as the other LEDs:
Snip the longer wire (anode) off the LED at the notch near the panel.
Cut one side of the resistor wire so it is about the same length as the nub of wire left on the LED.
Using grippers, align the short end of the resistor with the short wire on the LED.
Carefully solder these two together.
Do this for all three LED panels.

 

 

SECTION: Soldering LEDs
4.5. Connecting transillumination LED panels in parallel

Using ~10 cm pieces of white wire, solder together each anode (now with resistors), and each cathode. Hold them in place using shrink wrap.

 


 

Cut pieces of black wire and white wire about 0.3 meters long. Strip the ends of each. Solder the black wire to the cathode of the rightmost LED panel and solder the white wire to the anode of the same LED panel.

SECTION: Setting up the Arduino
5. The phenotype-o-mat uses an Arduino Nano Every to control its LEDs. This section covers how to set up the Arduino Nano Every and how to connect it to the LEDs.

If you haven't already, install the Arduino IDE.
SECTION: Setting up the Arduino
5.1. Loading the firmware

Connect the Arduino Nano Every to your computer via USB.
From our GitHub repo, download "arduino_control_through_serial.ino" and "serial_io.ino."
Open both of these files in the Arduino IDE and upload them to your device.

 


SECTION: Setting up the Arduino
5.2. Connecting LEDs to the Arduino

Bundle the wires together with heat shrink to keep them in place. Solder the wires from the LEDs to the Arduino in the following positions:

 

 

Place the Arduino Nano Every in the 3D-printed case and carefully feed the wires through. Connect the Arduino Nano Every to your computer via USB.

 

SECTION: Installing Spinnaker SDK
6. To use the FLIR BlackFly camera, you need to make a free account and download the Spinnaker Software Development Kit.

 


 

 

SECTION: Attaching hardware and 3D-printed components
7. Add the aluminum extrusion to the base

Now that your LEDs are wired together, you can assemble the phenotype-o-mat. Start by inserting the aluminum extrusion and fastening it to the plate-holder base with M3 thumbscrews.

 

SECTION: Attaching hardware and 3D-printed components
7.1. Attaching the transillumination LEDs to the plate-holder base

Carefully insert the LED panels face up, making sure not to scrape them on the edge on the way in.

 


 


SECTION: Attaching hardware and 3D-printed components
7.2. Attaching the camera arm

Slide the safety stop into place about 1/4 of the way from the base of the aluminum extrusion. Fasten it in place with the thumb screws.
Slide the camera arm into place and fasten it to the aluminum extrusion. You'll adjust it to the exact height once you attach the camera.

 

SECTION: Attaching hardware and 3D-printed components
7.3. Attaching the wired PCB ring and diffuser with the mount clip

Separate the mount clip and snap it in place on the microscope stand. 
Feed the PCB ring (now wired with LEDs) into the upper clip slot from the bottom, making sure there's room for the connected wires.
Feed the LED diffuser ring into the lower clip slot from the bottom.

 

 

 

SECTION: Attaching hardware and 3D-printed components
7.4. Attaching the camera mount and securing the camera

Loosen the knobs on the camera holder and place the camera mount with the gap facing toward the neck of the stand.
Make sure the camera is plugged into your computer via USB. Turn on the live view of the camera using the SpinView Software. Put a test plate on the base to note the camera's orientation and focus. Turn the camera so the USB cable is on the right or left side. This will give a "landscape" view of the plate. Adjust the height of the camera arm as needed.
Once you have the right focus and orientation, tighten the screws around the camera hole to keep the BlackFly camera in place. Make sure there's enough loose wire above the camera to prevent tension from moving the camera out of position.

 


 

SECTION: Attaching hardware and 3D-printed components
7.5. Attaching the filter slider

Insert the filter slider into the camera mount and use the visual indicators on the top to ensure the filter is directly underneath the camera lens.

 

SECTION: Attaching hardware and 3D-printed components
7.6. Building a dark box

Using black duct tape and black corrugated plastic sheets, create a dark box to house the phenotype-o-mat while assays run. Create a door using either a blackout curtain or by folding corrugated plastic.

 

We've also designed 3D-printable brackets if you'd like to give your box more structure, but they only fit 4 mm thick pieces of corrugated plastic, and duct tape is still required to fill in the gaps. You can use a piece of blackout curtain and velcro to make a door and magnets to secure the curtain to the sides of the box.

 

If you decide to print the dark box brackets, you'll need to print:
2× bottom_back_mag_bracket.stl
2× bottom_front_mag_bracket.stl
2× top_back_brackets.stl
1× top_left_bracket.stl
1× top_right_bracket.stl

SECTION: Running an assay
8. To run an assay on the phenotype-o-mat, clone our GitHub repo onto your computer, then run the script from your command line. Create a new directory to save the output files into.

To write your own assays, refer to the instructions posted in our GitHub.

 

SECTION: Overview
1. This protocol describes how to make a phenotype-o-mat in a step-by-step fashion. We've also included an overview figure below as an additional guide. It could be useful to print out as a reference as you work.