Biophysical & physicochemical methods for analyzing plants in vivo and in situ:y g p

Analysis of Photosynthesis BiophysicsAnalysis of Photosynthesis Biophysics via optical spectroscopy

f i l- a few important examples

Hendrik Küpper, visit to Třeboň and České Budějovice in April 2013

Pigment Quantification in Extracts:Modern UV/VIS-Spectroscopic Methodp p

Principle: 1) UV/VIS-Spectra are transferred into

th ti ti ll d “GPS t ”mathematic equations, so-called “GPS spectra” (published database currently contains 54 absorption spectra and 16 fluorescence spectra)spectra).

2) Before extraction, tissues/cells are frozen in liquid nitrogen and then freeze-dried. Afterwards, pigments are extracted in 100% acetone (forpigments are extracted in 100% acetone (for phycobiliprotein extraction from cyanobacteria, this step is followed by re-drying and extraction in 1x PBS)in 1x PBS).

3) A sum of the GPS spectra is then fitted to the measured spectrum of the extract. This fitting includes an automatic correction of base lineincludes an automatic correction of base line drift and wavelength inaccuracy of the spectrometer as well a residual turbidity and water content of the sample.p

Method of deconvolution: Küpper H, Seibert S, Aravind P (2007) Analytical Chemistry 79, 7611-7627

Measurement of in vivo / in situ-UV/VIS-Spectra(non-imaging)

Why in vivo / in situ?--> direct correlation with physiological parameters possible--> no extraction artefacts no extraction artefacts--> measurement on single cells possible--> high time resolution when measuring kinetics

Disadvantages compared to measurements of extracts:> many overlapping bands of the same pigment due to protein binding--> many overlapping bands of the same pigment due to protein binding

--> bands very broad--> extinctions coefficients in vivo usually unknown --> usually no absolute quantificationquantification

Example of the Application of in vivo-Absorption Spectra:Formation of Cu-Chl during Cu- stressg

Elodea canadensis

Ectocarpus siliculosus

Antithamnion plumula

Küpper H, Küpper F, Spiller M (1998) Photosynthesis Research 58, 125-33

Küpper H, Šetlík I, Spiller M, Küpper FC, Prášil O (2002) Journal of Phycology 38(3), 429-441

Analysis of in vivo-Spectra: Deconvolution

al d

ensi

tyO

ptic

a

Wavelength (nm)Wavelength (nm)

Purification of Trichodesmium phycobiliproteinsfor deconvoluting spectrally resolved in vivo fluorescence

kinetics and absorption spectrakinetics and absorption spectraPhycourobilin

isoforms Diazotrophic cell

eld

Phycourobilin isoforms

m)

basic fluorescence yield Fce

qua

ntum

yie

Phycobiliprotein purification + characterisation: Küpper H,

Andresen E, Wiegert S, Šimek M Leitenmaier B Šetlík I

isoforms (II)

axim

um)

o re

d m

axim

um

yield F0

ive

fluor

esce

nc

Method of deconvolution:

M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Phycoerythrinisoforms

alis

ed to

red

ma

(nor

mal

ised

to

eld

Rel

ati

Method of deconvolution:Küpper H, Seibert S, Aravind P

(2007) Analytical Chemistry 79, 7611-7627

Phycocyaninisoforms

scen

ce (n

orm

a

Fluo

resc

ence

PSII activity Fv

ce q

uant

um y

ie

Allophycocyanin

Fluo

res

ve fl

uore

scen

cR

elat

i

Necessary for energy transfer: stable S1-state

S2

i t t i

S1

intersystem crossing

i t t iT1

h·νb ti

intersystem crossing EET

photochemistry

h·νabsorption

absorptionfl

intersystem crossingintersystem crossing

phosphoresscence

S0

fluorescence

Chlorophyll

Necessary for energy transfer: Overlap of emission/absorption bands

M g - C h l a A b so rp t io n i n A c et o n M g - C h l a F lu o re sz en z in A c et o n

Abs

orpt

ion

A

5 5 0 6 0 0 6 5 0 7 0 0 7 5 0W e ll e n l ä n g e / n m

Measurement of in vivo chlorophyll fluorescence kinetics

Why?The quantum yield of in vivo chlorophyll fluorescence depends on a q y p y pcompetition for excitons between photochemistry (including electron transport after PSII via feedback), thermal relaxation ("nonphotochemical quenching") and fluorescence( nonphotochemical quenching ) and fluorescence.--> The fluorescence quantum yield and especially its change in response to changes in actinic irradiance allows a detailed

t f h t t II f ti d th th it lit f llassessment of photosystem II function und thus the vitality of a cell, tissue, plant or even ecosystem.

Examples of Applications- biophysical investigations of mechanisms of photosynthesisbiophysical investigations of mechanisms of photosynthesis- studies of the effects of abiotic and biotic stress on plants- ecophysiological studies

fruit quality assessment- fruit quality assessment

The basis for measurement of photosynthesis via fluorescence kinetics: competition for the S1 excited state

S2

i t t i

S1

intersystem crossing

intersystem crossingT1

h·νb ti

photochemistry

h·νabsorption

absorptionfl

intersystem crossingintersystem crossing

phosphoresscence

S0

fluorescence

Chlorophyll

Chlorophyll-Fluoreszenz

Wichtigste Symbole in der Chlorophyll-Fluoreszenzmessung

Biophysical measurements in vivo with temporal, spatial and spectral resolution: the Fluorescence Kinetic Microscope

dichroic mirrordetection dichroic mirror

single lens, lens system

filter (neutral, var. PC-controlled, long pass, short pass)

b/w measuring CCD camera

detection module

double TV-adapter

iris aperture

light source (LED)

mirrors (flat, concave), switching mirror

Fiberoptics-adapter for spectrometer

double TV-tube mot w. 2 outputs

adapter

( , ), g

beam unifying prism

shutter (manual, computer controlled)

Eyepiece

C-mount adapter for Mot Filter wheel

Mot. Filter

mot. filter & reflector cube

colour photo CCD camera Mot. Filter wheel

microscope

Mot. Filter wheelobjective

objectmicroscope stand

transmitted light sourcecondenser

excitation module

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74

Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4), 553-570

Fluorescence kinetic microscopyMethods of data processing

Method 1: images of fluorescence parametersMethod 1: images of fluorescence parameters

To obtain images of fluorescence parameters, frames within the relevantframes within the relevant time periods are selected and the necessary mathematical operations are

False colour image of Fm Chl fl l l t d f

False colour map of Fv/Fm, showing th diff i thi t

performed on every pixel.

fluorescence calculated from fluorescence kinetic film

the differences in this parameter over the entire image.

Method 2: kinetics of selected areas (objects)To obtain kinetic traces, the ,relevant regions are selected on a captured frame or parameter image. Th ki ti f ll i lThe kinetics of all pixels within the selected areas are averaged.

Manual selection of objects for kinetic analysis.

Fluorescence induction of selected objects, showing all differences in kinetics for representative cells.

Cd-stressed Thlaspi caerulescensImages of PS II activity parameters

CC

Cu

Cd

Fluorescence yield Efficiency of PS II Light saturation Electron flow through yduring Fm Fv/Fm Fm/Fp

gPS II during actinic irradiance (Fm'-Ft)/Fm'

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74

Spatial heterogeneity of photosynthetic oscillationsover the leaf surface

I t fl i i i (F ) th hit b t 100Insets: fluorescence emission images (Fp); the white bar represents 100 µm

Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens:images of PSII activity parameters

Image of Fm of an unstressed mature leaf

Image of Fm of a leaf stressed with 50µM Cd2, showing bright cells

Image of Fm of the same sample as on the left, lower magnification

Image of Fv/Fm of the same sample as above showing the

Image of Fv/Fm of the same sample Image of Fv/Fm of the same sample as on the left b t ithas above, showing the

homogeneously high photosynthtic activity of a healthy leaf of this plant

as above, showing the low photosynthtic activity of the bright cells

sample as on the left, but with lower magnification

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74

Cd-stress and acclimation in T. caerulescens & T. fendleri:histograms of Fv/Fm

20

30

T. caerulescens

Col

20

T. fendleri

ol

A

0

10

20C

ontro

20 D0

10

Con

tro

20 B

10

20 D

Stre

ssed

10

20 B

Str

esse

d

0

10

20 E

mat

ing

0

0Accl

im

20 F

ed

0

10

Acc

limat

e

0.0 0.2 0.4 0.6 0.8 1.0 Fv / Fm

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74

Spectrally resolved fluorescence kinetic parameters (II)

Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74

Spectrally resolved fluorescence kinetic parameters (I)

bright IIbright I

FluorescenceAbsorption

bright I

ores

cenc

e

bright InormallowFo activelowFo inactive

bsor

ptio

n

normallow Fo

FluoA

b

450 500 550 600 650 700 750 800Wavelength / nmCar

450 500 550 600 650 700 750Wavelength / nm

PUB = Phycourobilin

PC = Phyco-cyanin

PE = Phyco-erythrin Chl

RC (Chl)

APC =Allo-

Phyco-cyanin

Purification of Trichodesmium phycobiliproteinsfor deconvoluting spectrally resolved in vivo fluorescence

kinetics and absorption spectrakinetics and absorption spectraPhycourobilin

isoforms Diazotrophic cell

eld

Phycourobilin isoforms)

basic fluorescence yield Fce

qua

ntum

yie

Phycobiliprotein purification + characterisation: Küpper H,

Andresen E, Wiegert S, Šimek M Leitenmaier B Šetlík I

isoforms (II)

axim

um)

red

max

imum

) yield F0

ive

fluor

esce

nc

Method of deconvolution:

M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Phycoerythrinisoforms

alis

ed to

red

ma

norm

alis

ed to

eld

Rel

ati

Method of deconvolution:Küpper H, Seibert S, Aravind P

(2007) Analytical Chemistry 79, 7611-7627

Phycocyaninisoforms

scen

ce (n

orm

a

Abs

orba

nce

(

PSII activity Fv

ce q

uant

um y

ie

Allophycocyanin

Fluo

res

ve fl

uore

scen

cR

elat

i

Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual

h bili t iphycobiliproteins

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Prerequisites for FluorescenceResonanceEnergyTransfer (FRET)

Sequence of uncoupling events in a „bright II cell“„ g

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Light limitation: acclimation by reversible phycobiliprotein coupling: basic fluorescence yield F0

relative frequency

Andresen E, Lohscheider J, Šetlikova E, Adamska I, Šimek M, Küpper H (2010) New Phytologist 185, 173-188

Measurement of fast events, e.g. primary charge separation

Diode arrayarray

Iris

Diffraction grating

Sample cell

From: Berera R_vanGrondelle R_Kennis JTM_Ultrafast transient absorption spectroscopy_PhotosynthRes101_105-118

Measurement of primary charge separation

nge

ptio

n ch

anA

bsor

p

From: Lawlor DW (1990) Thieme, Stuttgart, 377S

Wavelength (nm)

Photosystem II reaction centre: steps of electron transfer to be analysed with thermoluminescence measurements to be analysed with thermoluminescence measurements

Thermoluminescence measurement

From: Rappaport F, Lavergne J (2009) Thermoluminescence theory. PhotosRes101, 205-216

Thermoluminescence measurement

From: Ducruet JM_Vass I_2009_Thermoluminescence experimental_PhotosRes101_195-204

Thermoluminescence measurement: parameters

From: Ducruet JM, Vass I (2009) Thermoluminescence experimental. PhotosRes101, 195-204

Thermoluminescence measurement: example

normal mildly dehydrated afterglow results from a heat-induced electron transfer fromstroma reductants to QB in the S2/3QB centers

B band is downshifted indicating a dark-stable acidic pH of lumenindicating a dark-stable acidic pH of lumen

From: Ducruet JM, Vass I (2009) Thermoluminescence experimental. PhotosRes101, 195-204

Decisive for measuring LIVING cells:keep the sample in physiological conditions!

medium inlet medium

tl tglass window (0.17 mm thick); sample

keep the sample in physiological conditions!

outletis placed between this window and the layer of cellophan

o-rings ( ili bb ) l f ll h(silicon rubber) layer of cellophan

Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4), 553-570

Decisive for measuring LIVING cells:don’t apply too much light!don t apply too much light!

Lens Light fielddiameter

Measuringirradiance

Actinicirradiance

Saturatingirradiance

[mm] [µmol m-2 s-1] [µmol m-2 s-1] [µmol m-2 s-1]

6 3 /0 20 2 90 0 006 686 524 6.3×/0.20 2.90 0.006 686 524 16×/0.40 1.06 0.026 2835 2167 25×/0.63 0.67 0.075 8295 6332 40×/0.95 0.38 0.200 22058 16904 63×/0.95 0.23 0.270 30311 23218100×/1.30 0.16 0.270 29441 22546

Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4), 553-570

Decisive for measuring LIVING cells:in order to be able to work with low light:

choose a suitable objective

All slides of my lectures can be downloaded

ffrom my workgroup homepage www.uni-konstanz.de Department of Biology Workgroups Küpper lab,

or directlyyhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html