Table 1 Measurement times required per sample for each of the R _dark techniques assessed

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