Team:UPenn/Engineering
ENGINEERING
The device hardware consists of two printed circuit boards (PCBs) sandwiching a standard 96-well clear-bottom plate. The top PCB is a modified OptoPlate-96 (Bugaj and Lim, 2019), and it consists of a 600nm used to take optical density measurements, a blue LED used to stimulate the light-dependent proteins, and in later iterations, a red LED for exciting red light optogenetic systems. The bottom PCB is referred to as the PlateReader and consists of a photodiode used to measure light intensity and a UV LED used to excite our fluorescent protein reporter, mAmetrine (Drobizhev, M., et al., 2011).
This device will be able to stimulate cells expressing optogenetically controllable protein circuits while monitoring the circuits output via fluorescence as well as cell growth through optical density in a 96-well plate format. Therefore the user would be able to run up to 96 different illumination patterns while obtaining live readout of fluorescent protein production and cell growth without disrupting the experiment in a high throughput manner.
In a typical experiment, there will be an optogenetically-responsive sample placed in each well of the 96-well plate. Blue light will control the optogenetic tool, and the combination of the OD LED, excitation LED, and photodiode will allow for real-time measurements of sample OD and fluorescence. The output of their optogenetic pathways may also be measured and observed. Thus, the OptoReader has the ability to stimulate and measure cells simultaneously.
OPTOPLATE
EXPERIMENTS
PLATEREADER
OptoPlate
The OptoPlate consists of 96 blue and red LEDs arranged in a 96-well plate format that allows for dual stimulation of blue and red light sensitive optogenetic tools (Bugaj and Lim, 2019). The OptoPlate was our impetus to create the Plate Reader and overall the OptoReader. We also fitted the Optoplate with a 600nm LED for measuring optical density.
Plate Reader
The PlateReader, consists of 96 UV LEDs and 96 photodiodes arranged in a 96-well plate format. The UV LED emits at a wavelength of 395 nm, and therefore allows for excitation of the fluorescent protein we use in our experiments, mAmetrine.
The photodiodes are light sensors that generate a current in response to photons allowing us to read a voltage accross a resistor. The voltage measured is proportional to the intensity of the light hitting the photodiode which allows us to measure protein fluorescence.
Each UV LED on the plate is connected to one of four LED drivers. An LED driver provides the electricity needed to power an LED. A single driver is able to control 24 different LEDs. Therefore the entire plate requires four drivers to supply power to each of the 96 LEDs. An LED driver has the ability to regulate the current and voltage that is supplied to the LED. This ensures that the LED has a consistent voltage applied to it so that its light does not flicker. Additionally, the drivers functions to regulate the amount of current that is applied to the LED. This allows for the LEDs to have their light intensity customized which will prove useful in optogenetic experiments. Each LED driver is connected to a capacitor and a resistor in order to to facilitate a steady current flowing into the driver.
Each photodiode is connected to one of six multiplexers. Each multiplexer can take on 16 photodiodes, thus requiring six multiplexers to power the entire array of photodiodes. A multiplexer takes in many inputs and choose which to send to the Arduino. As a result, the multiplexers can take a reading from many different photodiodes and come out with a single output. The entire plate is controlled by an Arduino Micro, a microcontroller, connected via USB to the Arduino interface on a computer.
Adaptors
In order to ensure that the two PCBs can successfully and snugly fit the 96-well plate in between them, two adaptors were designed and 3D printed to secured the PlateReader and the OptoPlate sandwiching a 96-well plate.
The bottom adaptor is secured to the PlateReader by screws and magnets, and it snaps into the bottom of the 96-well plate. This adaptor ensures that the 96-well plate does not shake on top of the PlateReader.
The top adaptor acts as more of a casing. It is secured onto the OptoPlate with screws and surrounds the 96-well plate. A small wall extends from the top adaptor into each well of the 96-well plate in order to ensure no spillage of liquid or light occurs between adjacent wells. A separator that covers half of each well and includes a small pinhole. The small hole allows red light to pass through into the well in a more focused manner. This ensures that there is no extra scattering of red light that would impact any optical density readings.
Filters
In order to ensure that the photodiode does not read the intensities of unwanted wavelengths, two filtering methods were used. The first is a physical filter, a thin film of acrylic that acts as a long-pass filter with a cutoff at 560 nm. Thus, since the stimulation of the fluorescent protein occurs at 395nm, very little of the light from the excitation LED should penetrate the filter to reach the photodiode. Additionally, the photodiode also has its own spectrum of light sensitivity. The photodiode acts as a band-pass filter that reads wavelengths between 480nm-660nm. This ensures that the photodiode is able to read light from the red LED (~600 nm) and the fluorescence (~525 nm). The photodiode also blocks out light from the excitation LED and the stimulation LED used to stimulate optogenetic tools.
Our software package includes device control as well as a Graphical User Interface (GUI) programmed in Python and C++ (Arduino)
Our Graphical User Interface (GUI) allows a user to easily set measurement intervals and optogenetic stimulation intensity and intervals for each well of a 96 well plate.
Software Architecture
The user inputs their protocol preferences in the GUI. Via serial communication, the protocols will be sent to the correct Arduino board to control the Blue, Red, and UV LEDs at the specified times.
The link to the iGEM Judging Release for our software package is found HERE
Why the Optoreader?
The OptoReader was designed, engineered, and tested to overcome current research barrier using optogenetics and increase the depth and types of experiments that can be carried out. Our team engineered an economical device that allows for high-throughput optogenetic experiments through simultaneous fluorescence and optical density readings and optogenetic stimulation in a 96-well plate format. The OptoReader consists of two printed circuit boards (PCB) that sandwich a 96-well plate. The top board is made up of an array of 96 LEDs used for opto-tool stimulation and 96 LEDs used for optical density measurements. The bottom PCB is made of a similar array of 96 LEDs and 96 photodiodes/light sensors that stimulate fluorescence and measure light intensity respectively. With a user-friendly graphical user interface, the OptoReader’s 96-well format allows for a high-throughput experimental design where the user can apply up to 96 different patterns.
References
1. Drobizhev, M., et al., Two-photon absorption properties of fluorescent proteins. Nat Methods, 2011. 8(5): p. 393-9.
2. Bugaj, L.J. and W.A. Lim, High-throughput multicolor optogenetics in microwell plates. Nat Protoc, 2019. 14(7): p. 2205-2228.
