Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment...

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Biophysical and physicochemical methods for analyzing plants in vivo and in situ: UV/VIS-Spectroscopy from pigment analysis from pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2013

Transcript of Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment...

Page 1: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Biophysical and physicochemical methods for analyzing plants in vivo and in situ:

UV/VIS-Spectroscopy from pigment analysisfrom pigment analysis

to quantification of mRNA

Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2013

Page 2: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence Principle, example: Chlorophyll

S2

p , p p y

i t l i

S1

internal conversion (= heat emission)

i t t iT1

h·νb ti

intersystem crossing

h·νabsorption

absorptionfl

internal conversioninternal conversion

phosphorescence

S0

fluorescence

Chlorophyll

Page 3: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 4: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 5: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 6: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 7: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Imaging in vivo-VIS-Spectroscopy: Modern Methods of Fluorescence Microscopypy

Important prereqisites and facts how to keep your cells alive while being measured aperture vs. light capture efficiency t t correct measurement overlap / interference of signals

Methods--> separation of chromophores--> FRET--> measurement of physiological parameters with fluorescent dyes--> FRAP

FCS--> FCS--> QISH--> fluorescent proteins

Page 8: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 9: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

Page 10: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Aperture

numerical Aperture NA = I * sinus q

I = refractive index of the mediumq = half opening angle of the objectiveq = half opening angle of the objective

Page 11: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

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

choose a suitable objective

Page 12: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Decisive for quantification:don’t overexpose!don t overexpose!

Page 13: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Decisive for quantification: correct calibration of the detectorcorrect calibration of the detector

Model: y = ax + b100

Model: y ax + b

Levenberg-Marquardt, statistical weighting

= 0.10176

alue 10

0.10176

a = 10.261 ± 0.157

b = -0.039 ± 0.069

grey

va

Pix

el

1

0 1 1 10Irradiance [%]

0.1 1 10

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

Page 14: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Important for interpreting fluorescence signals:Overlap of absorption / emission bandsp p

Page 15: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Preliminary tests with GFP in young leaves of Arabidopsis thalianaArabidopsis thaliana

Epidermis

40 μm

Mesophyll

Fluorescence observed through GFP filterset

NON-transformed plant...

All the signal was AUTOFLUORESCENCE

Page 16: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Use of overlapping Abs/Em-Bands for FluorescenceResonanceEnergyTransfer (FRET)

Page 17: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Prerequisites for FluorescenceResonanceEnergyTransfer (FRET)

Page 18: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes Principle

219

onm

ent 3

4, 2

08-

Transmitted light:

information about structure and cell typeC

ell a

nd E

nviro

and cell type

H, (

2011

)Pla

nt

aier

B, K

üppe

r H

dye fluorescence:

metal measurement

Leite

nm

Leitenmaier B, Küpper H (2011) Plant Cell & Environment 34, 208-219

Page 19: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes Types of dyes

organic dyes

f• Already available for many metals with many different binding and fluorescence characteristics

• Many dyes cell permeable

From: www.Invitrogen.com

nanoparticles

• new development, reliability and applicability not yet shown

• So far not cell permeable• So far not cell permeable

From: Méallet-Renault R, Hérault A, Vachon JJ, Pansu RB, Amigoni-Gerbier S, Larpent C, 2006, PhotochemPhotobiolSci 5, 300 - 310

Page 20: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes types of response

From: He CL et al 2006 AnalytSci 22 1547-

fluorescence quenchingconstant absorption

From: He CL et al., 2006, AnalytSci 22, 1547-

Page 21: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes types of response

From: www Invitrogen comFrom: www.Invitrogen.com

fluorescence turn – onconstant absorption

Page 22: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes types of response

From: www Invitrogen comFrom: www.Invitrogen.com

fluorescence constantratiometric absorption

Page 23: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes types of response

From: Chang CJ Jaworski J Nolan EM Sheng M Lippard SJ 2004 PNAS101 1129 34

ratiometric fluorescenceconstant absorption

From: Chang CJ, Jaworski J, Nolan EM, Sheng M, Lippard SJ, 2004, PNAS101, 1129-34

Page 24: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence of metal specific fluorescent dyes specificity

From: www.Invitrogen.com

Page 25: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Examples of non-quantitative applications:Animal cells

10 µM ZnAF-2F DA–loaded rat hippocampal slices

HeLa cells loaded with 50 μM Zn2+/pyrithione and 10 μM ZS5

rat neurons loaded with 100 μM Cu2+ & stained with 5 μM CS1

HEK-293T cellstreated with 1 μM MG1-AM and

5-day-old zebrafish treated with 50 μM of a Hg2+-selective dye

DC cells treated with a Cd2+-selective fluorophore (5 μM) and

From: Chang CJ, Jaworski J, Nolan EM, Sheng M, Lippard SJ, 2004, PNAS101, 1129-34

treated with 1 μM MG1-AM and exposed to 20 µM Hg2+

50 μM of a Hg -selective dye and 50 μM Hg2+

selective fluorophore (5 μM) and 5 μM Cd2+

Page 26: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

(3) UV/VIS fluorescence(a) Metal specific fluorescent dyes

calibration

Leitenmaier B, Küpper H, (2011) Plant Cell and Environment 34, 208-219

Page 27: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Quantitative measurement using metal-selective fluorescent dyes:Cd-uptake kinetics in Thlaspi caerulescens protoplastsp p p p

based on calibrated Rhod5N-fluorescence

Leitenmaier B, Küpper H (2011) Plant Cell & Environment 34, 208-219

Page 28: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Advantage of metal dyes under physiological conditions:correlation between metabolic activity and metal accumulation

Fv/Fm Fluorescence of Cd-dye

transient heterogeneity of mesophyll activity during period of Cd-g pinduced stress

correlates with transient heterogeneity of Cd-accumulation

in Thlaspi caerulescens!in Thlaspi caerulescens!

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

Page 29: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Analysis of molecule mobility:FluorescenceRecoveryAfterPhotobleaching (FRAP)FluorescenceRecoveryAfterPhotobleaching (FRAP)

Page 30: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

FluorescenceCorrelation

SSpectroscopy(FCS)

Page 31: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Fluorescence Correlation

S (FCS) IISpectroscopy (FCS) II

Page 32: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Fluorescence Correlation Spectroscopy (FCS) III

--> info about molecular info about molecular concentration, brightness, diffusion,

and chemical kinetics

Page 33: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Quantitative mRNA in situ hybridisation (QISH):overview of the method

cut out

max. 2x5 mm

vacuum infiltrate with alkaline fixation solution

extract pigments and dehydrate

extract hydrophobic compounds

rehydrate

digest proteins

postfixate

hybridise with

postfixate

quantify record images in CLSM extract and hybridise with fluorescent

oligonucleotides

quantify record images in CLSMquench background

Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1), 159-187

Page 34: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Analysis of metal transporter gene expression via a novel method for quantitative in situ hybridisation

Ch i i f h h d ff f i iCharacteristics of the method: effects of tissue optics

Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1), 159-187

Page 35: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Regulation of ZNT1 transcription analysed by quantitative mRNA in situ hybridisation (QISH)

in a non-hyperaccumulating and a hyperaccumulating Thlaspi species 10 µM Zn2+ Thlaspi caerulescens 10 µM Zn2+ Thlaspi arvense 1 µM Zn2+ Thlaspi arvense

0.4

0.5R

NA

)µ p

0.3

NA

) / c

(18s

rR

0.1

0.2

c(ZN

T1 m

RN

ophy

llhlo

emsh

eath

ophy

lle c

ells

y cell

sd c

ells

0.0

spon

gy m

eso ph

bund

le sh

palis

ade m

eso

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iderm

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ard

Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1), 159-187

epide

rmep

ide e

Page 36: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Qualitative Observation of Transcription&Translation in vivo via Fluorescent Proteins

transform Agrobacterium with the constructspromoter

Construct vectors for plant transformation

transfrom plants by agrobacterium infection (floral dip with or without

vacuum infiltration)

EYFP

vacuum infiltration)

germinate seeds of transformed plants on selective medium

select healthy (resistant) seedlings

(e.g. agar containing Kanamycin)

y ( ) g

select for YFP expression

prepare tissue pieces or whole mountsquantify record images in CLSM

Page 37: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

35S promoter in young leaves of Arabidopsis thaliana:epidermisp

Trichome base, epidermal cells and stoma Trichome

Overlays of green autofluorescence, red (chlorophyll) autofluorescence and yellow YFP fluorescence

Page 38: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

35S promoter in young leaves of Arabidopsis thaliana:mesophyllp y

Clone with high YFP expression Clone with medium YFP expression

Overlays of green autofluorescence, red (chlorophyll) autofluorescence and yellow YFP fluorescence

Page 39: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

Comparison of our in situ hybridisation method

i h GFP/YFP/D R d/with promoter-GFP/YFP/DsRed/... constructs

In situ hybridisation Fluorescent proteins

- Easy cellular quantification because whole cells are labelled

- Quantification on a cellular level difficult because only the narrow ring of cytoplasm is labelled

M i ( h l l t) b ti d- No macroscopic (whole plant) quantification possible because of diffusion limits

- Low background fluorescence because

- Macroscopic (whole plant) observation and quantification easily possible with fluorescence measuring camera (so far only tested with GFP)

- High background fluorescence because allgchlorophyll, carotenoids, flavonoids and many

further fluorescent compounds are extracted

- No direct comparison of gene expression with

- High background fluorescence because all autofluorescent compounds are present in the samples- Direct comparison of gene expression with physiological parameters (photosynthesisphysiology because samples are fixed (dead)

- Very fast: Ordering the fluorescently labelled oligonucleotides takes 1-2 weeks, the

physiological parameters (photosynthesis, electrophysiology) possible because samples are alive - Very time-consuming because of the cloning,

hybridisation procedure itself takes 3 days

- All plants can be analysed ( Thlaspi work)

transformation and plant growth/selection steps;

- The plant has to be transformed ( Arabidopsis)

- The gene sequence has to be known - The promoter has to be clonedThe gene sequence has to be known The promoter has to be cloned

Küpper H, Seib LO, Sivaguru M, Hoekenga OA, Kochian LV (2007) The Plant Journal 50(1), 159-187

Page 40: Biophysical and physicochemical methods for analyzing ...€¦ · from pigment analysisfrom pigment analysis to quantification of mRNA Hendrik Küpper, Advanced Course on Bioinorganic

All slides of my lectures can be downloaded

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

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

and

on the ILIAS websiteon the ILIAS website