A new tool for embolism visualisation: The 3D X-ray microtomography Eric BADEL Hervé COCHARD
UMR PIAF, INRA-UBP 5 chemin de Beaulieu, Clermont Ferrand
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2D X-ray radiography Röntgen 1895
I0
X-ray source
e, µ I
I ln( ) = − µ e I0
I0
I
thin sample 2D detector
µ : X-ray absorption is a function of : - atomic components • integrative information throught the thickness - matter density • spatial resolution is a function of sample - incident x-ray energy thickness and tomographic components (X-ray spot size, detector…) ALIMENTATION AGRICULTURE ENVIRONNEMENT
2-D X-ray tomography Medical scanner (low resolution)
2D tomography (high resolution)
Microfocus X-ray source High resolution detector - ln ( I / I0 ) 0.10
0.08
0.06
0.04
0.02
0 0
θ=0
Supreme Council of Antiquities ALIMENTATION AGRICULTURE ENVIRONNEMENT
The 3D µtomography
1000 to 2000 projections for 360° rotation ALIMENTATION AGRICULTURE ENVIRONNEMENT
Few X-ray tools
Small lab device
Lab device
Synchrotrons lights
X-ray
polychromatic
polychromatic
monochromatic
Beam
divergent
divergent
parallel
Spatial resolution
5-10 microns
1 micron
0.3 micron
Max sample size
4-5 cm
10 cm
1 mm
File size
16 Go
32 Go
64 Go or more
Scan time
10 min to 1 hour
10 min to 1 hour
1 s to few minutes
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The synchrotron lights
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The synchrotron facilities vs lab devices • very high x-ray intensity • fast scan time • the sample chamber is huge (but the scanned area is not larger than for the lab tools)
• access after a proposal • short time experiments • huge data to manage (1-4 To /j) • very tiring
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3D hydraulic network observation
New information about the vessels network ALIMENTATION AGRICULTURE ENVIRONNEMENT
Embolism measurements r 0 0 . 5
1
CAVITRON
Vulnerability curve
P50
Cochard et al 2005
Increasing tension and measurement of the loss of conductivity The big issue: not available for long vessels species ALIMENTATION AGRICULTURE ENVIRONNEMENT
Embolism observation and measurments Pinus sylvestris
Douglas 200 µm
Walnut tree 100 µm
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What did we learn about embolism process thanks to X-ray tomography? Few experiments using synchrotron radiations or our Nanotom (Clermont-Ferrand)
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Experiments using synchrotron light and living trees
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Experiments using synchrotron light and living trees light stimulation, fan, root stress => tension in hydraulic conduits
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Live embolism spreading observation (poplar tree)
3H Loss of conductivity (%)
10 min / scan using synchrotron radiation (Swiss Light Source)
3H
3H
3H
2
Poiseuille’s law
y=100/(1+exp(a*(x-b))), r =0.993 a=-4.50, b=1.80
100 75 50 25 0
0
1
2
3
Pressure (MPa)
3H
3H
3H
3H
Live embolism spreading observation (oak tree) 3H
3H
Cavitron X-ray images
Loss of conductivity (%)
100 2
y=100/(1+exp(a*(x-b))), r =0.89 a=-1.2, b=4.3 80
60
40
20
0
1
2
3
4
Pressure (MPa)
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5
6
Laurus: long vessels species -1 MPa
-3 MPa
100
100% embolized
Hacke & Sperry 2003 Dehydration Trifilo Salleo et al 2014a & Lo Gullo 1993 Trifilo Salleo et al 2014b et al 2000 Salleo & Gullo 1993 Salleo Hacke et al 1996 & Lo Sperry 2003 Salleo et Tyree et al 1998 Trifilo et al al 2000 2014a Salleo & Lo Gullo 1993 Hacke & Sperry 2003 Hacke Trifilo & Sperry 2003 et al al 2014b Salleo et 2000 et al 1996 2014a Salleo Trifilo et al 2004 Salleo Hacke et & al Sperry 2003 Air injection Trifilo etalal1998 2014b Salleo et al 2006 Tyree Trifilo et al 2014a Salleoet & al Lo1996 Gullo 1993 et Salleo Salleo et al 2009 Hacke 2003 Trifilo et Sperry al 2000 2014b Salleoet& Tyree 1998 Cochard 2002et al Salleo al 2004 Salleo 1996 2003 Hacke et & al Sperry Hacke & Sperry 2003 dehydration Salleo etetal alal1998 2006 Tyree et Trifilo 2014a Salleo et al 2004 centrifugation Salleo et 20092003 Hacke &etal Sperry Trifiloet al2006 2014b Salleo al Cochard 2002 Salleo et et al al 2009 1996 Salleo et al 2004 Salleo dehydration Tyree et 2002 al 1998 Salleo 2006 Cochard centrifugation Centrifugation Hacke & Sperry Salleo et al 2009 2003 dehydration Salleo et 2002 al 2004 Cochard centrifugation Salleo et al 2006 dehydration Salleo et al 2009 centrifugation Cochard 2002 dehydration centrifugation
100 80100 100
Percent xylem embolism Percent xylem embolism Percent Percent xylem xylem embolism embolism Percent xylem embolism
-6 MPa
Salleo & Lo Gullo 1993
Indirect Salleo et methods al 2000
80 100 60 80 80 60 80 40 60 60 40 60 20 40 Direct micro-CT 40 20 40 0 20 20 -6 -5 -4 -3 -2 -1 0 0 Xylem pressure, MPa 20 0 -6 -5 -4 -3 -2 -1 0 0 -6 -5Xylem -4 pressure, -3 -2and-1Badel 0 , submitted) MPa (Cochard, Delzon
-6
MPa -5Xylem -4 pressure, -3 -2
-1
0
Long vessel species , with big vessels do not-6show ‘R’ shaped MPa -5Xylem -4 pressure, -3 -2 -1 0 Xylem pressure, MPa vulnerability curve and can be very resistant! 0
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Embolism start Douglas
Poplar
(Dalla Salda et al, unpublished)
At the annual ring level, embolism is not a random process!
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Embolism spreading -1.5 MPa
-2.5 MPa
? Data for modelling at the cell arrangement level
Torres, Badel , Cochard et al in prep)
At the cell level, lonely seeds of embolism start more or less randomly; then spreading occurs in radial direction. Question: how? ALIMENTATION AGRICULTURE ENVIRONNEMENT
Freeze/thaw induced embolism
Freezing box
Cryo-stat
15
AEcum (%)
80 60 40 20 0
0
-20° < T° < +5° 0
30000
60000
90000
-15 -30
Temperature (°C)
100
Acoustic recording during X-ray scan
-45 120000
Charra Vaskou, Badel, Charrier, Mayr , Ameglio, in prep)
Time (sec) Air temperature
Air temperature of control chamber
Stem temperature
AEcum
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Conclusion - Prospect • X-ray observation may over-estimate the embolism but never under-estimate it! •
Great opportunity to look inside living trees under stress
•
Visualization will help to understand the mechanism.
•
X-ray tomography is becoming a reference method for embolism evaluation!
•
Need to ensure the tree health is not impacted by the x-ray radiations
•
Need to improve the sample environments for stresses control (cold, heat, RH%, light)
•
We always want more : higher resolution with larger field of view and faster scans.
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Thank you
…and if you do not have a synchrotron, you’re welcome in Clermont-Ferrand
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detector
The spatial resolution X-ray spot
Warning: the spatial resolution given by the manufacturer does not indicate the blurring The spot size is cruxial
pixel resolution =
det ector resolution G
X-ray spot
Same resolutions (given by the manufacturer!!)
blurred pixel = spot size × G
Detector resolution scan time small spot low x-ray intensity ALIMENTATION AGRICULTURE ENVIRONNEMENT
Quantitative resolution 1 mm
170
1 mm
120
160
niveau de gris
niveau de gris
130
110
100
150 140 130
90
0
100
200
position (pixel)
300
120
0
100
200
position (pixel)
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300
Embolism visualisation Pinus needle
Olive tree leaf
Charra-Vaskou et al, 2012
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