Overview

A genetic oscillator was built in E.coil as the real part while an e-oscillator was simulated in a computer as the virtual part. To realize our idea, two issues should be taken into account, the approach these two parts interacting with each other and the method cells keeping alive for the long and real-time observation. Thus, an interface device is needed in order to settle them. For the first one, light was chose as the medium to combine the virtual part and the real part together. And the other, cells cultivated on a microfluidic chip proved to be helpful.

In our project, the communication between these two parts is mutual which means that we need a two-way interface device composed of Light Receiving part and Light Controlling part. For the former one, cells were cultivated on a microfluidic chip and observed by fluorescence microscope. And for the last one, light (LED beads) can be controlled by the computer through a Single Chip Micyoco. Besides, we have made a different device to achieve the phase 2 collaborated with Model and another to complete the test of light control system with Wet Lab. Most important of all are that these two devices all turned to work successfully.

Interface Device

As is listed above, the interface device aims at making the communication between the virtual part and real part possible and keep cells alive during long time observation. For the first one, light is chose to be the media because of its specialty of physics as well as ability of influencing organism. For the last one, based on former studies, cells cultivated on a microfluidic chip proved to be helpful. After all the matters have been solved, we should go on for the whole design of device. The first step of our project is the bidirectional influence between virtual and real so that connection between them can be divided into two parts as the diagram below, Light Receiving which is from the real part to the virtual part and Light Controlling which is the opposite of it.

Fig.1 The whole design of Interface Device which is made up of Light Receiving part and Light Controlling part.

1 Light Receiving part

In this part, we are working on keeping cells alive and making them well detected by computer. The design is that cells are cultivated on a microfluidic chip which is put on an inverted fluorescence microscope. The CCD camera connected with microscope can get real-time snapshots of observed cells and the computer can transfer them into data of fluorescence intensity. Eventually, the Light Receiving part have passed the oscillations of real part from cells to virtual part. And then, it can connect with the data processing procedure in the computer.

1.1 Microfluidic chip

Microfluidic chips (Lab-on-chip technologies) originated from analytical chemistry and adopt sophisticated technologies to make micro-channels about several square centimeters on chip. This technology integrates sample’s injection, separation and detection into a signal chip.

Why do we choose microfluidic chip among so many tools?

The design of our chip

*1. Two kinds of chambers

There are two kinds of chambers, one is a square room with 30nm height while another looks like a swimming pool with 15nm depth and 30nm width. After cells and medium are injected into micro-channels, parts of cells can be trapped in chambers proliferating freely and chambers will be full of cells in about 3 hours while superfluous cells are squeezed out chambers.

Fig.2 The blueprint of our microfluidic chip with two kinds of chambers.

*2. Three inlets and one outlet

As is shown below, our microfluidic chip is made up of three media inlets and a waste outlet while the green arrow points to waste outlet and the blue arrow points to media inlet. The media inlet 1, 2, 3 is used to inject fresh medium IPTG and arabinose respectively and the outlet is for discharging the effluent and injecting bacterial solution.

Fig.3 The left one is the actual image of our microfluidic chip and the presentation of its inlets and outlet (The green arrow stands for waste outlet while the blue are for media inlets).and the other is the view of our chip with flowing cells observated by industrial microscope.

1.2 Inverted fluorescence microscope

For the real-time observation of fluorescence intensity, a fluorescence microscope is needed. In the same time, for a clear vision of a single chamber, the chip have to be observed with 100x oil lens which means that the lens may almost touch the chip. Thus, with four transparent pipes on the chip, it seems to be inconvenient by normal fluorescence microscope but inverted one is perfect.

Inverted fluorescence microscope is made of fluorescent accessories and inverted microscope, and mainly used in the observation of living organisms. As a result of these living detected objects are placed in a culture dish or bottles, it requires a long working distance between objective lens and condenser lens for direct observation. Therefore, the position of the objective lens, condenser and the light source are reversed. And also, a CCD camera is connected with the microscope so that we can get real-time photos and even a video during observation.

As for the expensive price of using the high-precision Confocal Laser Scanning Microscope in our school, we decide to use it only for final result. To be available for normal use, we have bought a small industrial microscope (400 x) and designed it into our device for early observation.

Fig.4 The side and bottom view of this small industrial microscope (400 x).

2 Light Controlling part

In this part, we are working on passing the information from the virtual part to the real part. A microcontroller and several LED light beads are used to achieve this goal. Specifically speaking, after analyzing two oscillators, the computer will change parameters in its own oscillator as well as the illumination intensity of LED as you can see in the modeling part. And then, a USB line is used to transfer the command from the computer to a microcontroller which are able to control the illumination intensity of LED directly. Based on all of these, the oscillator of cells can be controlled which means that the information flowing from the virtual part to the real part achieved. The core of the Light Controlling part is programs on the microcontroller to communicate with both the computer and LED light beads.

2.1 SCM (Single Chip Microcomputer)

SCM is a kind of integrated circuit chips and have the ability to integrate central processor CPU, RAM, RAM, read-only memory ROM, several I/O mouths, interrupt system and timer/counter function (may include display driver circuit, pulse width modulation circuit, analog multiplexer and A/D converter circuit) into a piece of silicon consisting of a small but perfect microcomputer system. So, we use it to transform signal from PC to LED beads.

One of our team numbers is good at Python and another is C, so the former one chose pyboard while the other chose MSC51 to realize goals in the same time. Different as the tools we use are, the design of them have rare diversity.

Fig.5 The photos of STC 51 chip and Pyboard.

2.2 LED beads

Considering that light control system in our engineering bacteria is sensitive to specific wavelengths of light, we buy LED beads of red light in 660nm and 740nm. The relationship between light intensity and current of them is close to linear which means that their current in some sense can represent for their light intensity.

Fig.6 LED beads of red light in 660nm.

Fig.7 Parameters of LED beads which can directly reveal that the relationship between electric current and intensity is liner. （from the Internet）

What’s more, as for the fact that the maximum working current of our SCT51 chip is only about 20mA but 350mA for these LED beads, we add a ULN2003 chip and a 10 uf electrolytic capacitor on the circuit to reduce the burden of it after precise calculation .

Fig.8 ULN2003 chip and a 10 uf electrolytic capacitor is used to prevent.

3 Current progress in interface device

3.1 Program of Interface Device

The goals that this part are focus on are as following.

• Communicate with MATLAB and read its data about every 2 minutes.

• Transfer the command from computer to SCM.

• Control LED beads by SCM.

Till now, we have completed the program of this part. For STC 51 chip, we divided it into two parts, one is the PC program written in C# and another is for the SCM written in C. MATLAB program are separated into several parts so that they can be inset in C# and be called easily. And then, PC program send a set of data that STC 51 need before next adjustment. Finally, STC 51 chip would know the specific data of current even in every 0.01 second and follow it.

Device demo video

3.2 Hardware of Interface Device

According to the requirements of microfluidic chip, inverted fluorescence microscope and SCM, like being horizontal and smaller than the volume of microscope table, we designed a device to place our components. And then, we printed it in FDM with 3D Print Technology. It’s suitable with other parts of our device and strong enough for supporting the microfluidic chip.

3.3 Lab Test Device

Fig.9 a. The microfluidic chip b. Our experiment space c. The vision of microscope

As you can see in the Wet Lab part, we need to test whether the light control system could work. Hence, Wet Lab need several LED beads with different light intensity in the same time. So, three I/O pins connect with three LED beads and SCM assigns different values of PWM to them.

Usage procedures:

● Inject bacterial culture solution with 3 hours’ incubation into media inlet by syringe pump.

● Use industrial microscope (400 x) for real-time detecting and photos.

● Put all above in a drying oven to maintain 37 ℃.

● Computer is lined with SCM though a USB line.

We used the device to observe cells and have got satisfying results.

Fig.10 The vision of Lab Test.

Mixed-reality of physics

As you can see at the Project page, to simplify the goal, we have divided our final goal into three phases. Compared the purpose between phase 1 and 2, phase 2 can be regarded as just putting out one of the two oscillators in computer into LED while others are nearly the same. In the same time, the target as well as a program for the interface device at phase 2 and 3 make almost no difference. So based on the successful result of phase 1 and completed phase 3, phase 2 seem to be achieved easily. The phase 2 is called Mixed- reality of physics which means that the coupling between the virtual part in computer and the real part of physics. The assumption is that the oscillators of computer and the LED can react with each other and end up in highly coupling.

Design principles

As for this part, we intend to construct a perfect circle.

Fig.1 the diagram of our design

At the beginning, the e-oscillator and the physical oscillator work independently. A period of time later, the camera start to detect the light intensity of LED beads and send the data to a computer. After quick analysis of these two oscillators, the computer would change the parameters of oscillation functions and send changed new data to a single chip. Finally, the status of LED would be uploaded after the command of a single chip. And then, go on this loop again and again till the Mixed-reality sate achieved.

Device

Based on the design principles above, we constructed a device as following.

a. The whole view of our device

b. Details of pyboard c. Details of LED beads d. Details of camera

Fig.2 The composition of our device and details of every parts.

Results

The program of computer is in MATLAB while it’s Python in Pyboard. And the results turned to be inspiring that the whole design worked successfully, including interface device, programs and the simulation in computer.

The phase 2 has completed and this device can be applied to the phase 3 directly. Once this simple camera is replaced by a fluorescence microscope with cells cultured on a microfluidic chips, the device of the phase 3 would be done and are possible to make Mixed-reality state come true. Thus, it’s the prototype of MR. Cell and has great potential in a wide range of the future biological research.