Inspiration

In our project, several schemes were proposed to demonstrate the functionality of our genetic circuit in 96-well plates. The top option would be to observe the cells under different light under a fluorescence microscope or to measure their fluorescence intensity on a microplate reader. However, the microplate reader cannot fully capture the result, and the microscope is restricted to a single well instead of the whole plate. What's more, the oscillation period of the repressilator is rather long, which means that the observation time span would be so long that multiple and regular manual captures are not practical. Additionally, long-time operation could possibly damage the laser generators or CCD of these instruments. To overcome these issues, a specific piece of equipment was designed.

Aim

1. To build an independent and affordable device for the observation of colonies or 96-well plates.
2. Automatically capture and save digital pictures of colonies according to the pattern we designed.
3. Add LED lights and filters for fluorescence observation.

Parts

Part	CNY/Unit	Details
Raspberry Pi 3B +	¥319.0
Power Supply for LED	¥72.0	Output
Stabilize-voltage Regulator Module	¥40.0
10 W LEDs (Blue)	¥15.0	OSRAM LE B Q8WP
10 W LEDs (Green)	¥15.0	OSRAM LE CG Q8WP
10 W LEDs (Ultra White)	¥15.0	OSRAM LE UW Q8WP
LED radiator	¥0.6
5 M Camera Module	¥138.8	two
USB cables	¥2.5
wires	¥30.0
8GB micro SD card	¥40.0
500nm Emission Filter, Φ=11mm	¥120.0	For GFP observation
620nm Emission Filter, Φ=11mm	¥120.0	For RFP observation
3D-printed Shell	Free	Material: XY
4-Panel Solid State Relay	¥65.0

Construction

The whole device consists of three parts, and a 3D-printed scaffold is applied to hold them together.


First, the power supply converts 220V AC into 15V DC, and transfers it to the voltage-stabilization module, which can be finely adjusted in terms of both output voltage and amperage. The stable out-put ensures that the LEDs work under the same power conditions or generate excitation light with the same intensity.

Second, the embedded system Raspberry Pi 3B+ is applied in this work, controlling the LED lights in conjunction with the cameras. A simple Python program was written to set the wanted photo-shooting procedure. Figure 2 shows the scheme of the program.

The third part comprises the optics, including high-power LEDs and 5-Megapixel camera modules with specific filters. The high-power LEDs provide stable high-intensity excitation light for fluores-cence. Because there is no second port on the Raspberry Pi 3B+ board for its official camera module, we purchased 2 USB2.0 cameras instead, to enable two fluorescence observation channels. Each camera is equipped with a filter corresponding to the emission wavelength of one fluorescent protein. The associated parameters are attached as hyperlinks in the parts sheet above.

Problems during construction

System freezing

At first, we used the unfinished device to shoot pictures of culture dishes and wanted to do some fine-tuning. Unexpectedly, the device stuck randomly during the shooting procedure with no warning signs. The only way to save the photo procedure was to reboot the Raspberry Pi and continue with the shooting. After several attempts, we found it's wiser to use a high-speed TF card rather than a normal TF card to avoid this error.

Unstable white balance

In our case, pictures of culture dishes were taken every 5-30 minutes using light of a single color. It would be ideal to maintain the same white balance in a series of pictures under white-light set-tings. While testing the device, some pictures of a series looked greenish. This might be a flaw of the module itself. However, this camera module is not a universal part, and we have no access to its detailed instructions and parameters. It is also difficult to find a pattern to eliminate those less-than-perfect photos. After several tests, we suggested a long light expose before shooting as a solution. Lowering the shooting frequency or continually running a couple of shooting processes might help stabilize the camera.

General test of the device and comments

In order to test the robustness and utility of the system in monitoring large objects, we had the pleasure to invite several researchers to test and comment on our device.

Researcher 1: It's such a convenient device! It acts as a security camera. With this tiny thing, I'm able to have my bacteria tracked with nothing left. Hope that it can be connected to a PC and manipulated remotely.

Researcher 2: Generally, we use a fluorescence microscope for single-cell observations and a plate reader for quantitative measurements. This gives us a solution for general and long-term observation.

Researcher 3: I'm willing to use this equipment for trial tests to avoid unnecessary usage of expensive precision instruments.

More things we can do

In order to observe the functionality of our clock-repressilator for the iGEM project, we made this device and fine-tuned it. We hope to refine this simple and convenient device and assist other iGEMers as well as other researchers. We aim to improve the camera characteristics and optic system functions while keeping it affordable.



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