TITLE: Measuring PPFD on Algal Shaker
AUTHORS: Jakub Nedbal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The illuminated orbital shaker features a glowing growing area. The illumination is not entirely homogeneous. It is brightest near the centers of the LEDs and becomes dimmer closer to the edges. The light brightness of the light in the glowing area is dependent on the LEDs, their spacing, the distance between the LEDs and the transparent platform, the material of the anti-slip mat, among other parameters. To ensure consistency and reproducibility between experiments, the brightness can be measured and the light output of the illuminator calibrated. Quantum detector is used to measure the photosynthetic photon flux density (PPFD). The measurement described in this protocol is conducted for all current settings on the LED controller and in 12 different locations across the glowing area. It is done both in air and inside an Erlenmeyer flask containing water, to best capture the actual conditions experienced by the microalgae cultures.</div></div>

[STEPS]
?. [Preparation]
Switch off the algal shakerClear out any culture flasks from the algal shaker.Move it onto a bench.
?. [Preparation]
Work in a dark room. Turn all lights in the room off. Keep them off during the measurements.
?. [Preparation]
Connect the Walz probe to the Li-COR Light Meter.Set the Li-COR Light Meter calibration to measure in air (-274.3). This value is specific to the particular probe and will be different for other probes. The value in the calibration data for specifically for the utilized probe must be used.
?. [Measurement]
Place the probe on a random spot on top of the illuminated shaker platform and record the value.Hold the probe vertically upright and keep your hand as far away from the illuminator as possible to minimize effect of light scattered from the hand and sleeve.Repeat this in twelve different random places. Find a random location by doing it with eyes closed or looking away to avoid personal bias.
?. [Measurement]
Repeat the above step for all eight LED current settings.
?. [Measurement]
Record all PPFD measurements in this table:  ABCDEFGHI1RepeatLED Current Setting2Trickle·12345631425364758697108119121013111412
ABCDEFGHI1RepeatLED Current Setting2Trickle·12345631425364758697108119121013111412
?. [Measurement]
Set the Li-COR Light Meter calibration to measure in water (-471.9). This value is specific to the particular probe and will be different for other probes. The value in the calibration data for specifically for the utilized probe must be used.
?. [Measurement]
Place a 100 ml Erlenmeyer flask onto the illuminated shaker platform.
?. [Measurement]
Place the probe into the flask fix the flask mouth with some cotton wool, replicating the plug normally used in the culture. Try to keep the probe vertical.Record the value.Move the flask to another random spot, repeating the measurement in twelve different places. Find a random location by doing it with eyes closed or looking away to avoid personal bias.
?. [Measurement]
Repeat the above step for all eight LED current settings.
?. [Measurement]
Record all PPFD measurements in this table:  ABCDEFGHI1RepeatLED Current Setting2Trickle·12345631425364758697108119121013111412
ABCDEFGHI1RepeatLED Current Setting2Trickle·12345631425364758697108119121013111412
?. [References]
ReferencesLI-250A Instruction Manual​​US-SQS/L Instruction ManualPPFD project: Code to present the results of the PPFD measurement in a graphical format.UnivarScatter project: MATLAB® code by Manuel Lera Ramírez used to produce univariate scatter plots.​
?. [Analysis and Data Presentation]
Download the MATLAB® code to analyze the PPFD measurement from GitHub:github.com/jnedbal/PPFDThe project directory contains the following files:  AB1FileDescription2Air.pdfGraphical representation of the PPFD results measured in air in PDF format3Air.pngGraphical representation of the PPFD results measured in air in PNG format4LICENSEBSD 2-Clause license5PPFD.xlsxTable with PPFD measurement results. Update the table with your own measurements6PPFD.mMATLAB® code to analyse the measurement data in PPFD.xlsx table. Requires UnivarScater.7Water.pdfGraphical representation of the PPFD results measured in water in PDF format8Water.pngGraphical representation of the PPFD results measured in water in PNG format
AB1FileDescription2Air.pdfGraphical representation of the PPFD results measured in air in PDF format3Air.pngGraphical representation of the PPFD results measured in air in PNG format4LICENSEBSD 2-Clause license5PPFD.xlsxTable with PPFD measurement results. Update the table with your own measurements6PPFD.mMATLAB® code to analyse the measurement data in PPFD.xlsx table. Requires UnivarScater.7Water.pdfGraphical representation of the PPFD results measured in water in PDF format8Water.pngGraphical representation of the PPFD results measured in water in PNG format
?. [Analysis and Data Presentation]
Update the PPFD.xlsx file downloaded in the above step with the actual measurement values obtained from your illuminated orbital shaker.
?. [Analysis and Data Presentation]
PPFD requires function UnivarScatter downloaded from Mathworks® File Exchange:https://uk.mathworks.com/matlabcentral/fileexchange/54243-univarscatterThe UnivarScatter project was written by Manuel Lera Ramírez.Place the files from the UnivarScatter project in the same folder as PPFD.m on your computer, or put it into another path, where MATLAB® can find it. You can use addpath function for that purpose. Without UnivarScatter, PPFD will throw an error.
?. [Analysis and Data Presentation]
Run PPFD in MATLAB®. The graphs will be produced according to the measurement values in PPFD.xlsx spreadsheet.