MICROSPECTROFLUOROMETRY FOR LOCALIZATION
OF COMPOUNDS IN LEAF TISSUES
G. Agati ([email protected]), P. Matteini
Istituto di Fisica Applicata “Nello Carrara” – CNR, Sesto (FI)
M. Tattini, L. Traversi
Istituto per la Valorizzazione del Legno e delle Specie Arboree - CNR FI
Z. Cerovic, A. Cartelat
Groupe photosynthese et teledetection LURE/CNRS, Orsay France
F. Bussotti, E. Gravano, C. Tani
Dipartimento di Biologia Vegetale, UNIFI
supported by:
CNR Target Project on Biotechnology;
CNR-CNRS bilateral cooperation project
n. 11409;
Universita’ degli Studi di Firenze
Fluorescence from endogenous compounds in leaf tissues
Localization of compounds in leaf tissues is an important tool for
14000
optimizing fluorescence
monitoring of vegetation
(blue-green and red-Chl
fluorescence signatures)
FLIDAR
Adapted from Magritte, Les tables de la loi, 1961
2)
10000
Blue-green fluorescence
(several fluorophores)
8000
6000
4000
understanding mechanisms of response to stress conditions
3)
•chlorophyll-a
12000
punctual
fluorometer
fluorescence (a.u.)
1)
Red fluorescence
(single fluorophore)
l exc 365 nm, leaf cross section
2000
0
understanding the functional roles of particular classes of
compounds (polyphenols)
380
480
580
680
wavelength (nm)
780
Phenylpropanoids
Cofactors
Others
•hydroxycinnamates
•coumarins
•flavonoids
•pyridine
nucleotides
•flavins
•alkaloids
•quinones
APPARATUS AND METHODS
Acquisition and analysis of
fluorescence spectra
Gaussian deconvolution
for band separation and
quantification
Sequential acquisition of
fluorescence images at the
spectral bands of interest
Imaging by a chargecoupled device (CCD)
camera with narrow-pass
(10 nm) optical filters for
band separation
Image elaboration by a suitable
computation function
Digital imaging allows
suitable image elaboration:
math operations
background removing
flat-field correction
false color
representation
recombination
Epifluorescence
microscope
Dichroic
mirror
Multichannel
spectral
analyzer
Optical fiber
trichomes
Red
Blue
recombination
Exc.
lamp
Filter
wheel
470 nm (Dl =10 nm)
Interference
filter
Sample
Cooled
CCD
camera
Autofluorescence imaging in Triticum aestivum L. (wheat)
(lexc = 365 nm)
Mobile mirror
Bandpass
filter
400
500
600
700
Visualization of the
multispectral image
800
adaxial
epidermis
20 mm
680 nm (Dl =10 nm)
Chl and hydroxicinnamates
co-localization
Localization of flavonoids in leaf tissues of Phillyrea latifolia L.
1.2
fluorescence (a.u.)
to understand their functional role in the acclimation mechanisms
to excess light stress
shade leaf
1
sun leaf
0.8
0.6
comparison between sun and shade plants
lexc = 436 nm
lem = 470 nm
lem = 580 nm
0.4
Light regime
sun = 480 W/m2
shade = 70 W/m2
Flavonoid fluorescence must be induced
( e.g. by Narturstoff reagent)
lexc = 365 nm
Red
Blue
0.2
0
380
480
580
wavelength (nm)
680
merging
780
0.5
14000
0.4
diff. fluorescence (a.u.)
12000
10000
8000
SUN LEAF
6000
SHADE LEAF
a)
4000
2000
0
32
64 96 128 160
0
sun - shade
0.3
0.2
0.1
475
0
575
-0.1
-0.2
cell wall
cuticle
hydroxicinnamates
guard cells
sclerenchyma bands
-0.3
-0.4
-0.5
380
480
580
680
780
380
480
580
wavelength (nm)
680
780
Different contributions to leaf surface fluorescence
F580
sun
b)
adaxial
epidermis
shade
Studying the plant response to ozone stress in Acer pseudoplatanus L.
100 mm
F580 - k ·F470
c)
trichome
Fluorescence (a.u.)
20x10
3
Autofluorescence,
normal
damaged
mesophyll
15
546 nm
lexc = 436 nm, Dlem = 10 nm)
680 nm
Line Profile
120
lexc = 365 nm
10
100
5
500
600
700
Intensity
80
0
400
800
Wavelength (nm)
damaged l exc=436nm
40
Suitable fluorescence image acquisition and elaboration permits to evidence the tissue
specific localization of flavonoids and their large difference between sun and shade leaves.
Fluorescence (a.u.)
4000
F580/F470
60
20
3000
damaged
l exc=365nm
0
2000
0
2-bands
1000
0
400
480
520
560
Wavelength (nm)
200
300
400
Distance (Pixel)
merging
normal l exc=365nm
440
100
600
640
Compound accumulation (yellow fluorescence) in ozone-damaged tissues