Table 1.

Measurement times required per sample for each of the R _dark techniques assessed

Technique	Step	Description	T (min)
Fluorophore	Calibration	Purge tubes of air using N2 gas or sodium dithionite	0.02–0.05
	Sample preparation	Dissect tissue (e.g. scalpel, scissors or leaf punch) and place in measuring tube	0.5–1
	Measurements	In general, slopes taken from 1 to 2.5-h. 186 samples per run. Note: more than 186 samples can be simultaneously measured but cycle time between O2 recordings will increase to >6-min, reducing resolution	0.8
	Total		1.3–1.9
O2-electrode	Calibration	Prepare and assemble electrodes, including application of membrane and electrode solution. Aerate calibration solutions and obtain zero and saturated O2 values after stabilisation of current	4–9
	Sample preparation	Dissect tissue and place inside cuvette and adjust plunger being careful not to introduce air pockets	1–2
	Measurements	Slopes taken after stabilisation of signal and before depletion of O2, usually within 10–40 min but dependent on sample	20–40
	Total		25–51
IRGA	Calibration	Change consumables (e.g. soda lime, desiccant, CO2 canister) and zero IRGA chambers	1–2
	Sample preparation	Select and clip measuring chamber onto leaf	0.5–1
	Measurements	Allow steady-state gas-exchange to be reached	10–15
	Total		11.5–18
MIMS	Calibration	Apply membrane and test membrane stability. Purge tube and inject known volumes of O2 and CO2. Record background consumption	5–10
	Sample preparation	Dissect tissue and place inside cuvette and air-seal cuvette	1–3
	Measurements	Allow signal to stabilise (usually 5 min) and record slope between 5 and 20 min	20
	Total		26–33

T (min) represents the estimated time it takes to measure a single sample in minutes. For example, if 20 samples can be measured without recalibration and it takes 20-min to calibrate, then the calibration T is 1-min