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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