Kinetic Imaging of Plant Chlorophyll Fluorescence

Experiment Kautsky and Hirsch (1931) irradiated a dark-adapted leaf with a blue light and observed it visually through a dark-red glass. Here is a high-tech presentation of what they saw: © Photon Systems Instruments, spol. s r.o., CZ

Chla fluorescence competes with photosynthesis for excitation energy S0S0 S1S1 S2S2 Chla 1 h blue fluorescence h NIR photosynthesis © Photon Systems Instruments, spol. s r.o., CZ

FluorCam of Photon Systems Instruments, Ltd. FluorCam was designed to measure heterogeneity of plant photosynthetic activity among species, mutant plants or within a single plant from the whole plant level down to individual cells. Localized biotic or abiotic stress is a typical factor causing heterogeneity in the plant activity that can be visualized by FluorCam. FluorCam measures fluorescence image transients of leaves, small plants, and of algal or cyanobacterial colonies. Typical size of an investigated object is smaller than 10  13 cm The measured fluorescence is excited by short measuring flashes. The transients are induced by a continuous actinic light and by plastoquinone- reducing pulses of strong irradiance. Typical output is a 2-dimensional map of F 0, F M, F V, F V /F M or of NPQ. © Photon Systems Instruments, spol. s r.o., CZ

Fluorescence QA-QA- 750 LED’s are on for  s Only few PSII RC’s are excited Yet, sufficient fluorescence emission is produced to capture an image Measuring flashes have little actinic effects © Photon Systems Instruments, spol. s r.o., CZ

QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- During the actinic light exposure, the continuous excitation keeps some of the PSII RC’s closed LEDs are on for seconds to minutes Actinic light is causing fluorescence induction © Photon Systems Instruments, spol. s r.o., CZ

F0F0 F PEAK from F 0 with open PSII RC’s to F PEAK with mostly closed PSII RC’s In fluorescence, the actinic light elicits in plants the Kautsky effect of fluorescence induction. QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- © Photon Systems Instruments, spol. s r.o., CZ

Kautsky effect in a diuron-inhibited leaf © Photon Systems Instruments, spol. s r.o., CZ DCMU

Before the pulse During the pulse, PSII RC’s are closed by a transient reduction of the plastoquinone pool. The shutter of the halogen lamp is open typically for 1s QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- PQ-reducing super pulse © Photon Systems Instruments, spol. s r.o., CZ

Fluorescence before the pulse F0F0 Open PSII reaction centers The closure of all PS RC’s is reflected by a transient from F 0 to F M. Fluorescence at the end of the pulseFMFM QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- QA-QA- Fluorescence in PQ-reducing super pulse. © Photon Systems Instruments, spol. s r.o., CZ

F0F0 FMFM FVFV FSFS FM’FM’ Pixel-to-pixel arithmetic image operations

Color photograph Fluorescence F M image Chlorophyll fluorescence from ripe lemon fruits

Color photograph Fluorescence images F0F0 FVFV FMFM F V /F M Heterogeneous lemon pigmentation

Color photograph Fluorescence images F0F0 FMFM F V /F M FVFV Post-harvest lemon damage

Green mold infection of lemons Color photographs Fluorescence Time after infection 0 h0 h48 h66 h84 h

Histograms reveal parameters of the highest contrast FMFM F0F0 FVFV

The mold-infected peel tissue contains detached antenna complexes healthy infected

Phytotoxin response visualized by fluorescence Sinapis alba 60 h, 2000 mg/l destruxin Brassica oleracea 60 h, mg/l destruxin 0.05 mg/l 0 mg/l 0.5mg/l 50mg/l 500mg/l

Mutant selection

High-light stress sensitivity F V /F M 2 © Photon Systems Instruments, spol. s r.o., CZ

Saturating Pulses: ON Young Leaf Old Leaf A B C Actinic Light: OFF Leaf development

Field operation

50  m F V / F M FSFS F S – F 0 Microscopic kinetic fluorescence imaging diatoms Elodea chloroplasts Average

Heterocyst formation in cyanobacteria Transmittance F M fluorescence