Description

Open-JIP is an open-source chlorophyll a fluorometer used to quantify photosynthetic phenotypes in terrestrial plants and microalgae. It is controlled via a Teensy microcontroller and features highly customisable electrical components and a 3D-printed infrastructure.

OJIP

Open-JIP utilises a technique known as the fast polyphasic rise fluorescence (also known as OJIP). Steady irradiance is provided from an emitting LED, this excites photosynthetic antenna in the sample resulting in excess irradiance being emitted as photons of a longer wavelength (i.e. fluorescence). The emitted fluorescence, which contains information regarding the state of photosynthesis, is captured by a photodiode, amplified by the custom circuitry and stored by the Teensy microcontroller. This all happens in less than a second.

The transient captured by Open-JIP gives the user an indication of the state of photosynthesis in the subject organism to varying levels of complexity. If you would like to know more about the theory surrounding the measurement of chlorophyll a fluorescence, this link gives a basic introduction and resources for further reading.

Publication

The original device which used an Arduino Mega was published in Photosynthesis Research in 2019:

A guide to Open-JIP, a low-cost open-source chlorophyll fluorometer - Bates et al. 2019

If you would like to see what the device is capable of, you may be interested in this recent publication which uses Open-JIP as a flow-through chlorophyll a fluorometer connected a custom photobioreactor with the microalga Chlorella vulgaris.

Collaborations and custom architecture

If you would like to collaborate on a project, or would like to see if Open-JIP can be customised to suit your application, please email me at [email protected].

Disclaimer

Open-JIP requires some knowledge of electronics, programming and photosynthesis to operate. If you have problems with the device place an issue in the issues tab of this repository and I will do my best to address it as soon as possible.

USER MANUAL

Below contains the user manual for Open-JIP, if you have any questions there is a discussions tab in this github repository, where I will do my best to get back to you as soon as possible.

Contents

1. Introduction
   1. Specifications
   2. Getting started
      1. Downloading files
         1. Method 1 (New users)
         2. Method 2 (Experienced users / collaborators)
      2. Gathering materials
         1. Printed circuit board (PCB)
            1. Ordering a PCB
         2. 3D models
         3. Additonal parts
2. Firmware / Software
   1. Introduction
   2. Arduino IDE / Teensyduino
   3. Basic operations
      1. Basic functions
      2. Advanced functions
      3. Calibration functions
   4. Python control
   5. Advanced operations
      1. Change actinic LED intensity
      2. Change detection sensitivity
      3. Set analog reference voltage & Resolution
      4. Defaults
      5. Length of actinic pulse
3. Hardware
   1. Detection
      1. Gain adjustment
   2. Actinic Intensity

1. Introduction

Specifications

Feature	Value / Description
Actinic LED Colors	Blue 455 – 485 nm (466 nm), Green 517 – 555 nm (532 nm), Orange 583 – 600 nm (593 nm), Red 617 – 635 nm (626 nm).
Resolution	10 to 16-bit (optimised for 12-bit)
Sampling rate	8 μs at 12-bits
Operating voltage	15 V 1.2 A (Barrel plug)
Communications	USB (Serial)
Microcontroller	Teensy 3.5/3.6 or Teensy 4.1 (3.6 Recommended)
Actinic Intensity	Four intensities (Low, Mid, High, Very High)
Detection (Gain)	Four settings (Low, Mid, High, Very High)

Getting started

Downloading files

Method 1 (New users)

New users should navigate to the releases page to find the most recent (and stable) version of the device. You can download the latest release as a .zip file and this will contain all the files you need to get the device up and running.

The most recent version is v0.3.

When searching through files in this repository all relevant folders are labeled with a specific version. You will want to use files that match the version you are using.

Method 2 (Experienced users / collaborators)

If you want to contribute to the project or fork this directory feel free to do so, just know that I constantly update files here so they may not function as expected before release.

To clone this directory type this into your terminal / console.

git clone https://github.com/HarveyBates/Open-JIP.git

Gathering materials

Open-JIP can be built in an afternoon if you have all the equipment at hand. The folder "Bill of Materials" contains all the materials needed to build Open-JIP. This has been divided into two parts; (1) circuit board and (2) other.

In addition to the Bill of Materials you will need to have access to:

1. A soldering iron
2. Solder (Paste or wire variety)
3. A re-flow oven (not necessary but makes the circuit board easier to assemble)
4. A 3D-printer
5. A pair of wire strippers / cutters
6. Some tweezers

Printed circuit board (PCB)

In the Bill of Materials/Circuit Board folder you will find a table all the components needed to construct the Open-JIP circuit board. These components can be bought from common electronics suppliers such as digikey, LCSC or farnell etc. Links for each component is provided.

Note that most of the components are surface mount (SMD), this is due to their increased reliability and in my opinion its easier to assemble SMD compared to through-hole (THT) components.

Ordering a PCB

To order the circuit board I recommend using JLCPCB as they are cheap, easy and ship fast.

1. Open the folder titled Electronics
2. Navigate to your current version
3. In the subdirectory select the folder containing gerber and drl files
4. Compress this folder into a .zip file.
5. On the JLCPCB website drag and drop the .zip file into the order form.

The default settings are suitable.

3D Models

The 3D-models used for 3D-printing Open-JIP can be found in the Open-JIP/Hardware folder. Two file types are specified:

1. .stl can be directly imported into 3D-printing software such as Ultimaker-Cura or sent to a local 3D-printing manufacturer.
2. .STEP is the modifiable Open-JIP 3D-model and as such can be configured whichever way you want before being exported as a .stl file. You can use 3D modeling software to do this such as Fusion360 or Solidworks.

Additional parts

In addition to the above electronics you will need a few components such as a Teensy microcontroller, power supply, computer to use the device etc. A table of all these additional items can be found in the folder location Bill of Materials/Other.

2. Firmware / Software

Introduction

Open-JIP operates via a computers serial port (USB connection), the user sends commands in the form of strings that are read by the Teensy microcontroller. This results in the device taking a measurement or other tasks. You can do this manually though the Arduino IDE or by using scripts in various languages.

Arduino IDE / Teensyduino

To setup the Teensy (upload the firmware to the microcontroller) you will need both Arduino IDE and TeensyDuino if running on Linux or Windows, or just TeensyDuino if using a Mac. Instructions on how to do this is provided in the above links.

When first using Open-JIP:

1. Plug the Teensy into your computer
2. Open Arduino IDE
3. Select Tools>Board>Teensy 3.6 (may be in another part called Teensyduino)
4. Select Port>"Your Teensy Device"
5. File>Open the "Teensy.ino" file found in Firmware/v0.3/Teensy_3.6/Teensy_3.6.ino
6. Sketch>Upload to flash the file onto the Teensy

The provided Teensy.ino file will work as is, but if you wish to change settings in this file you will need to reupload the firmware to the Teensy again after changes have been made.

Basic Operations

To take a measurement open the serial monitor (in the Arduino IDE when the device is connected to your computer) (Tools>Serial Monitor) and type in the command MF (denoting measure fluorescence). This will (in the default configuration) take a one second measurement of chlorophyll a fluorescence and print the resulting measurements out into the serial monitor. You can copy and paste the data into excel in order to graph the transient.

There are a number of included functions that require the same method to operate:

Basic functions

1. MF - Measure fluorescence. Measure the chlorophyll a fluorescence signature of the organism of interest.
2. CP - Calculate parameters. Calculate and print out basic fluorescence parameters from the most recent OJIP curve.
3. CFo - Calibrate F_o (the minimum level fluoresence). Provides short flashes of illumination for calibrating the minimum level fluorescence. This command is useful if you want to know if your measurement is going to saturate (due to an overly concentrated sample) before taking a measurement.
4. An - Actinic intensity (irradiance of LED) where n is a number between 1 and 4 (lowest to highest).
5. Fn - Fluorescence gain (sensitivity of detection) where n is a number between 1 and 4 (lowest to highest).

Note - It is recommended that the minimum level of fluorescence (F_o) is greater than 0.3 V for the maximum stability of acquisitions.

Advanced functions

1. MJ - Measure J-step. Measures up to the J-step (~2 ms) and returns the data to the serial port.
2. MW - Measure wave. Measures up to the J-step multiple times with set time intervals between acquisitions.

Calibration functions

1. ML - Calibrate actinic intensity (measure light). Turns on the actinic LED for a period of three seconds to allow the intensity to be measured by a 4π light meter.
2. Cr Calibrate rise time of the actinic LED/amplifier combination. Provides some short (100us) flashes of illumination from the actinic LED to calibrate your setup using an external oscilloscope. Useful if you want to ensure your Fo value is accurate (i.e. Fo should be measured when the actinic LED and amplifier are stable (usually around 40us)). Note this is only nessesary if you alter the default electronics configuration (see below).

Python Control

Open-JIP comes with an external cross platform python script that allows users to take basic measurements, plot the data and sort it into a .csv file automatically. This program is located under External Control/Open-JIP.py.

Open-JIP.py uses plotly to plot the data and pyserial to access the serial port of your machine. You must have python installed to use this script. This program runs in the terminal / console. Additonally you need pip to install these requirements as follows:

cd Open-JIP/Software
pip3 install -r requirements.txt
python3 Open-JIP.py
# or (depending on python installation)
pip install -r requirements.txt
python Open-JIP.py

If you wish to change the output path of the .csv file you can do so by changing this string in Open-JIP.py:

fileName = "Open-JIP_Data.csv"  # Filename of output .csv file

Advanced operations

A major feature of Open-JIP is its customisability. In the firmware users can adjust:

1. The actinic LED intensity
2. The detection sensitivity
3. The analog reference voltage
4. Resolution of the device
5. Length of actinic pulse

This next section is going to be in reference to the files located within Firmware/v0.3/Teensy_3.6.

Change actinic LED intensity

In actinic.cpp the actinic LED intensity can be adjusted. There are four possible configurations, LOW, MID, HIGH, VERY HIGH. These can be selected by applying the values 1 - 4 (LOW - VERY HIGH) when switching on the actinic LED.

void Actinic::on(){
  switch(intensity){
    case 1:
      digitalWrite(actinicGain1, HIGH); // Low
    	break;
    case 2:
      digitalWrite(actinicGain2, HIGH); // Mid
      break;
    case 3:
      digitalWrite(actinicGain3, HIGH); // High
      break;
    case 4:
      digitalWrite(actinicGain4, HIGH); // Very high
      break;
  }
}

Note- Electrical components have intrinsic variability. If using identical devices ensure actinic intensity is similar using a PAR meter.

DO NOT - combine actinic LED intensities as this will result in undefined behaviour.

Change detection sensitivity

In sensitivity.cpp the detection sensitivity can be adjusted. There are four possible detection configurations, LOW, MID, HIGH, VERY HIGH (1 - 4). These are set before starting an exposure for stability and are therefore defined by the user.

void Sensitivity::define(int _gain){
    refresh();
    switch(_gain){
        case 1:
            // Lowest
            digitalWrite(fluoroGain1, HIGH); // Photosynthesis (OJIP)
            break;
        case 2:
            digitalWrite(fluoroGain2, HIGH); // Photosynthesis (OJIP)
            break;
        case 3:
            digitalWrite(fluoroGain3, HIGH); // Concentration only (not recommended for photosynthesis)
            break;
        case 4:
            // Highest
            digitalWrite(fluoroGain4, HIGH); // Concentration only (not recommended for photosynthesis)
            break;
    }
}

NOTE

Recent testing of the gain settings of Open-JIP has led to the conclusion that the current version of the device (v0.3) can only use gain 1 and 2 for photosynthesis (OJIP) measurements. Gain 3 and 4 should only be used for concentration estimates using the fluorescence intensity at ~120 μs.

DO NOT - combine gain settings as this will result in undefined behaviour.

Set analog reference voltage & Resolution

In fluorescence.cpp the reference voltage can be adjusted to either 3.3 or 1.1 volts when using a Teensy 3.6. The purpose of this is (1) the Teensy 3.6 (1.1 V) reference voltage is more stable than 3.3 V and (2) you can attain a higher resolution when measuring weak fluorescence signals.

void Fluorescence::set_reference_voltage(float voltage){
  // Sets and initialises the required reference voltage for measurements
  if(voltage == 3.3){
    analogReference(DEFAULT); // Set to 3.3 V
  }
  else if(voltage == 1.1){
    analogReference(INTERNAL1V1); // Set to 1.1 V
  }
  else{
    analogReference(DEFAULT); // Set to default (3.3 V) if unknown value is found
  }
  analogRead(readPin); // Initialise reference voltage
}

Note - This is usually done statically at the start of the program by adjusting the value of refVoltage to either 3.3 or 1.1 depending on your requirements.

The number of bits for each reading can be changed via this command in the void setup() function:

analogResolution(12); // 12-bit resolution

Note - Increasing the resolution decreases the sampling rate so if you want to get alot of data points around Fo I reccomend you use a 12 or 10-bit resolution.

If you use have a 12 bit-resolution with a reference voltage of 1.1V the finest sensivitiy of the device is 0.00027 V per-division (1.1V / 2¹²) at a 8 μs sampling rate.

Defaults

Upon startup, Open-JIP will revert to its default configuration. You can change this by altering the setup command in Teensy_3.6.ino:

void setup() {
  Serial.begin(115200); // Starting baud rate
  analogReadResolution(12); // Default resolution
  pinMode(13, OUTPUT); // Sets the microcontrollers LED pin as an output
  
  sensitivity.refresh(); // Switch off all sensitivity settings
  sensitivity.define(1); // Set a default sensitivity (1 - 4)

  actinic.off(); // Switch off all actinic settings
  actinic.define(1); // Set a default actinic irradiance (1 - 4)
}

Length of actinic pulse

In fluorescence.h the length of the actinic exposure (pulse) can be adjusted. The acquisition is done in two steps; (1) the fast measurments (readings every 8 μs) and (2) slow (readings every 1 ms). These arrays require different processing methods and are therefore stored seperately. Below gives you an idea of how the array lengths are calculated for the intial aqusitions and the converted output.

// Microsecond (micro) = readings every 8 us
static const unsigned int microLength = 1000; 
int microRead [microLength];
int milliRead[microLength];

// Millisecond (milli) = readings every 1 ms
static const unsigned int milliLength = 1000; 
int microTime[milliLength]; 
int milliTime[milliLength];

// Stores final array in converted form (i.e. Volts and Milliseconds)
float fluorescenceValues[microLength + milliLength];
float timeStamps[microLength + milliLength];

Note - If you are using the Open-JIP.py script you will need to change the number of data points that are read to match the length of fluorescenceValues:

timeStamps, fluorescenceValues = measure_fluorescence(2000) # Match with teensy script array length (default 2000)

3. Hardware

Open-JIP operates using a Teensy 3.6 microcontroller connected to a custom printed circuit board (PCB). The circuit is divided into two parts:

1. Detection - Responsible for detecting emitted fluorescence
2. Actinic intensity - Responsible for turning on and off the actinic LED

Detection

Fluorescence detection is achieved with the use of an operational amplifier (op-amp) connected as a transimpedance amplifier. The inverting input is connected to a photodiode, while the non-inverting input is connected to a voltage divider to provide a very small offset voltage. The concept of the offset voltage is that the amplifier (op-amp) will respond faster to changes in voltage.

Note- Resistor values may change depending on your application.

Gain adjustment

Four different sensitivities are provided though the use of a CMOS switch (74HC4066D) which has a range of different feedback resistors (1M, 2M, 3.3M and 3.9M). These resistors are all 0805 SMD. The gain is selected in the firmware of Open-JIP; however, if you wish to adjust the senstivity futher you can do so by changing one or more of these resistors.

Increasing the resistance of the feedback resistor will increase the sensitivty of detection.

Decreasing the resistance of the feedback resistor will decrese the sensitivty of detection.

Note

Feedback resistances above 2Mohm are not suitable for OJIP measurements in v0.3. Ideally the feedback resistor should be a small as possible to increase the response time of the device.

Note

If you begin to get a large overshoot occurring at the start of your measurements you may need to increase the capacitor value (C3) to 2pF - 5pF. This article may help you if you encounter this issue.

Actinic intensity

The actinic LED is controlled in the same way as the detection circuit i.e. Using a CMOS switch to controll its brightness. Again four levels of brightness can be selected in the firmware and are controlled by resistors (10K, 12K, 15K, 20k) connected to the base of the darlington transisitor (BD681). Adjusting the value of these resistors will result in the LED getting brighter or dimmer based on their resistance value.

Note- Resistor values may change depending on your application.

The input voltage (15 V) is filtered through a 12 V linear voltage regulator. This regulator ensures the actinic LED is stable of the measurement. A small capacitor (C7 - 1 μF) is placed across the LED's terminal to prevent overshoot. If you want the LED to respond faster you can reduce the value of this capacitor while using an oscilloscope to check the devices rise-time.

Increasing the resistors results in decreased actinic brightness.

Decreasing the resistance of R10 results in increased actinic brightness.

Note

Gate resistor values are dependent on a number of factors including manufacturer tolerances and LED color. Dialing in the resistor values to use in your device is an important consideration. Typically you will need a PAR meter to calibrate the devices brightness before use (if using for OJIP measurements). However, if access to a PAR meter is an issue, the shape of the OJIP curve can be an indicator of LED intensity. If the rise between Fo and Fj is sharp and rapid (<2ms), typically the device has enough brightness to saturate the electron transport chain. However, if the rise between Fo and Fj is gradual (>2ms), the brightness may not be bright enough.

Future of Open-JIP

Since the publication of Open-JIP a number of changes have been made to increase the devices capabilities. These include:

1. Adjustable fluorescence protocols

2. Ability to change detection sensitivity dynamically (though software)

3. Adjustment of actinic LED intensity dynamically (through software)

4. Improvements in resolution

5. Improvements in number of datapoints in each OJIP transient

Licence

The software in this work is licensed under GNU AFFERO GENERAL PUBLIC LICENSE Copyright (C) (2020) (Harvey Bates)

This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with this program. If not, see https://www.gnu.org/licenses/

For more information contact: [email protected]

The hardware (3D Models, Electronic Schematics) in this work is licensed under a Creative Commons Attribution-ShareAlike 3.0 Unported License.