imagerie biologique par spectrométrie de masse tof-sims et ...ipnimagerie biologique par...
TRANSCRIPT
12 juin 2012
Alain BRUNELLE
Centre de recherche de GifInstitut de Chimie des Substances Naturelles
Gif-sur-YvetteFrance
Imagerie biologique par spectrométrie de masse TOF-SIMS et MALDI-TOF
1er Workshop Equipex ANDROMEDE
Bio-Imaging and Mass Spectrometry
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 2
In vivo :
• X ray(absorption)
• PET (Positron Emission Tomography)
• MRI (Magnetic Resonance Imaging)
Ex vivo :
• Fluorescence (absorption/emission)
• Radio-labelling (emission)
• IR (absorption)
•AFM (Atomic Force Microscopy)…
All these techniques only target a few molecules, which have been selected a priori …whereas mass spectrometry allows the simultaneous analysis of a large number of moleculeswithout any a priori and with no labelling.
Focused beams of UV photons (MALDI) and/or cluster ions (SIMS) make possible to raster asurface
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE
Mass Spectrometry ImagingMALDI-TOF versus TOF-SIMS imaging ?
MS Images Ion density maps 1 acquisition = one image for
each peak and one spectrumper pixel…
Huge amount of data
3
Section mincecongelée
TOF MS
4000 6000 8000 10000
Spectre de masse [m/z]
Images MS
Ions ouPhotons
Section mincecongelée
TOF MS
4000 6000 8000 10000
Spectre de masse [m/z]
Images MS
Ions ouPhotons
Frozen section MS images
Ions orphotons
Mass spectrum (m/z)
M. Stoeckli, P. Chaurand, D.E. Hallahan, R.M. Caprioli, Nat. Med. 2001, 7, 493-496.
Mass spectrometry ? Fast Simultaneous information from
many compounds Large mass range High sensitivity
Applications: Human health Screening of modified molecular
repartitions in tissues Diagnostic of pathologies Study of drug distribution in
biological tissue duringtreatments
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE
Mass Spectrometry ImagingMALDI-TOF versus TOF-SIMS imaging ?
MS Images Ion density maps 1 acquisition = one image for
each peak and one spectrumper pixel…
Huge amount of data
4
Section mincecongelée
TOF MS
4000 6000 8000 10000
Spectre de masse [m/z]
Images MS
Ions ouPhotons
Section mincecongelée
TOF MS
4000 6000 8000 10000
Spectre de masse [m/z]
Images MS
Ions ouPhotons
Frozen section MS images
Ions orphotons
Mass spectrum (m/z)
M. Stoeckli, P. Chaurand, D.E. Hallahan, R.M. Caprioli, Nat. Med. 2001, 7, 493-496.
MALDI-TOF-TOF TOF-SIMS
Spatialresolution 50 µm 400 nm - 2 µm
SampleDehydrated,
Homogeneousmatrix coating
DehydratedNo fixationNo matrix
Mass range m/z > 200 m/z 1500
Compounds
Proteins,Peptides,Lipids,Drugs,
Metabolites,…
LipidsGlycosphingolipids
Cyclopeptides,Drugs,
Metabolites, Elements,…
Analyzer TOF-MS/MS TOF-MS
Lipids Lipids:
Various biological functions Major structural components of cell membranes Energy storage Antioxidants Cell signaling
Composition and distribution of lipids depend on: Cellular type Status of a group of cells Physiological conditions
Chemical imaging: anatomo-pathology lipophilic dyes
Poor specificity: all the lipids, or only a lipid family Mass spectrometry coupled to gas or liquid chromatography
Excellent for quantification and/or identification Preliminary extraction loss of the spatial information on tissue
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 5
Nile RedBlack Soudan
Excitation 450-500 nmEmission >528 nm
Excitation 515-560 nmEmission >590 nm
Apolar lipids Polar lipids
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE
TOF-SIMS imaging
TOF-SIMS mass spectrometers equippedwith liquid metal ion sources (Ga+ ions) allowing lateral resolution << 1 µm
Widely used for surface analysis in polymerindustry, metallurgy, semi-conductor
Until 2002-2004, very few applications in organic mass spectrometry because of the poor efficiency of Ga+ ion sources for intact ion emission above 100-200 and separationbetween different scientific communities
Radical change with the advent of cluster ions sources since 2002
6
D. Touboul, F. Halgand, A. Brunelle, R. Kersting,E. Tallarek, B. Hagenhoff, O. Laprévote
Anal. Chem. 2004, 76, 1550-1559D. Touboul, O. Laprévote, A. Brunelle
Curr. Opin. Chem. Biol. 2011, 15, 725-732
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 7
Increase the secondary ion emission with clusters
The impact of a polyatomic projectile is the unique way to bombard a very small area
simultaneously with several atoms
Deposition of a highenergy density veryclose to the surface
Non Linear Effects
Ga1+ Projectile
Ions or Neutrals
Ions or Neutrals
Bi3+ Projectile
M.Benguerba, A. Brunelle, S. Della-Negra, J. Depauw,H. Joret, Y. Le Beyec, M.G. Blain, E.A. Schweikert,
G. Ben Assayag, P. SudraudNucl. Instrum. Methods. Phys. Res. B 1991, 62, 8-22
A. Brunelle, S. Della-Negra, J. Depauw, D. Jacquet,Y. Le Beyec, M. Pautrat, Phys. Rev. A 2001, 63, 022902, 1-10
N. Wehbe, M. Fallavier, S. Della-Negra,J. Depauw, A. Brunelle, H.H. Andersen,
Nucl. Instrum. Methods. Phys. Res. B 2010, 268, 2596–2602
S. Bouneau, A. Brunelle, S. Della-Negra,J. Depauw, D. Jacquet, Y. Le Beyec,M. Pautrat, M. Fallavier, J.C. Poizat,H.H. Andersen,Phys. Rev. B 2002, 65, 144106 1-10
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 8
Increase the secondary ion emission with clusters
The impact of a polyatomic projectile is the unique way to bombard a very small area
simultaneously with several atoms
Deposition of a highenergy density veryclose to the surface
Non Linear Effects
Ga1+ Projectile
Ions or Neutrals
Ions or Neutrals
Bi3+ Projectile
Bi3+Bi1+ Images recorded at the edge of the corpus callosum on a rat brainsection256x256 pixels, 256x256 µm2
Pixel size 1x1 µm2
Same fluences 1012 ions.cm-2
Acquisition time : 4 and 10 minutes
D. Touboul, F. Kollmer, E. Niehuis, A. Brunelle, O. Laprévote,J. Am. Soc. Mass Spectrom. 2005, 16, 1608-1618
How to read an ion image
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE
Intensity bar
m/z value to which the
image corresponds
mc = max counts = numberof counts in the most
intense pixel
tc = total counts = total number of counts in the
image
1
mcImages of 256x256 pixels
= 65 536 pixels
= 65 536 mass spectra!!!
9
TOF-SIMS lipid imaging
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 10
D. Touboul, F. Kollmer, E. Niehuis, A. Brunelle, O. Laprévote, J. Am. Soc. Mass Spectrom. 2005, 16, 1608-1618
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 11
Sample preparation for tissue imaging
After dissection, fast freezing at -80°C Tissue is maintained with OCT glue 12-20 µm sections cut with a cryostat (-15 -20°C) Tissue sections are deposited directly onto a stainless steel plate,
a silicon wafer, or a glass plate Dehydrated under vacuum Fixed onto the standard sample holder
Tissue
Same method as for MALDI,with no matrix coating
MALDI Imaging
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 12
+ ++ + ++
Mixture between matrix and sample molecule(5000 for 1)
Co cristallization (solid sample)
Pulsed UV Laser (106 W.cm-2)
Desorption - Ionisation of sample molecules(molecular ions [M+H]+)
Analysis by mass spectrometry
Matrix Assisted Laser Desorption Ionization
a long work in methodology to obtain homogeneous and reproducible matrix
coatings, enabling analysis of the surface with a good sensistivity
HTX ImagingTM-Sprayer
MALDI Imaging: a long way since the beginning
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 13
"Image" of the m/z 7605 protein fragment from humanbuccal mucosa cells (cheek)
1997
• MALDI-TOF mass spectrometers with 2 kHz lasers• Data acquisition and processing software• Robots for matrix deposition• 50 µm resolution
Excellent method for in situ lipid analysis
Today
Rat brain sagittal sectionTM-Sprayer (HTX Imaging)
MALDI-TOF and TOF-SIMS Lipid imaging
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 14
H I J
K L M
(+)
N
D I : 0-250
m/z 283.1
F I : 0-1959
m/z 888.7I
E I : 0-200
m/z 303.2
(-)
A I : 0-40
m/z 780.7
B I : 0-38
m/z 796.8I
(+)
C I : 0-100
m/z 806.7
G
(-)
5 mm
O
Rat brainAdjacent sagittal sectionsMALDI pixel size: 50 µmSIMS pixel size: 87 µm
TOF-
SIM
SM
ALD
I-TO
F
F. Benabdellah, A. Seyer , L. Quinton, D. Touboul , A. Brunelle, O. Laprévote, Anal. Bioanal. Chem., 2010, 396, 151-162
Identification?
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 15
7 2 0 7 6 0 8 0 0 8 4 0 8 8 0 9 2 0 9 6 0 1 0 0 00
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
Inte
nsity
m /z
7 6 6 .6
7 2 8 .6
720 730 740 750 760 770 7800
20
40
60
80
766.6
Inte
nsity
m/z
728.6
723.6
m/z 728.6
Identification?
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 16
•Only glycerophospholipids•42 combinations:
•17 GPCho•9 GPSer•16 other
•Biological relevance:•even fatty acids
only (!) 19
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 17
Ion path in reflector mode
Laser path
MALDI TOF/TOF
4800 MALDI TOF/TOF Applied Biosystems
MALDI MS/MS: in-situ identification
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 18
0 100 200 300 400 500 600 7000
4000
8000
12000
16000
464.
3 (M
-C18
:1+H
2O)
446.
3 (M
-C18
:1)
436.
3 (M
-C20
:1+H
2O)
418.
3 (M
-C20
:1)
79.0
(PO
3- )97
.0 (H
2PO4- )
140
(pho
spho
etha
nola
min
e)
309.
3 (C
20:1
)
281.
2 (C
18:1
)
Inte
nsity
m/z
255.
2 (C
16:0
)
724 726 728 730 732 7340
40
80
120
Inte
nsity
728.56
PEDatabases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup
Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)
No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.
C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171
MALDI MS/MS: in-situ identification
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 19
0 100 200 300 400 500 600 7000
4000
8000
12000
16000
464.
3 (M
-C18
:1+H
2O)
446.
3 (M
-C18
:1)
436.
3 (M
-C20
:1+H
2O)
418.
3 (M
-C20
:1)
79.0
(PO
3- )
97.0
(H2P
O4- )
140
(pho
spho
etha
nola
min
e)
309.
3 (C
20:1
)
281.
2 (C
18:1
)
Inte
nsity
m/z
255.
2 (C
16:0
)
724 726 728 730 732 7340
40
80
120
Inte
nsity
728.56
PE 18:0p/18:1Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup
Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)
No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.
C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171
MALDI MS/MS: in-situ identification
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 20
0 100 200 300 400 500 600 7000
4000
8000
12000
16000
464.
3 (M
-C18
:1+H
2O)
446.
3 (M
-C18
:1)
436.
3 (M
-C20
:1+H
2O)
418.
3 (M
-C20
:1)
79.0
(PO
3- )
97.0
(H2P
O4- )
140
(pho
spho
etha
nola
min
e)
309.
3 (C
20:1
)
281.
2 (C
18:1
)
Inte
nsity
m/z
255.
2 (C
16:0
)
724 726 728 730 732 7340
40
80
120
Inte
nsity
728.56
PE 16:0p/20:1Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup
Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)
No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.
C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171
MALDI MS/MS: in-situ identification
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 21
0 100 200 300 400 500 600 7000
4000
8000
12000
16000
464.
3 (M
-C18
:1+H
2O)
446.
3 (M
-C18
:1)
436.
3 (M
-C20
:1+H
2O)
418.
3 (M
-C20
:1)
79.0
(PO
3- )
97.0
(H2P
O4- )
140
(pho
spho
etha
nola
min
e)
309.
3 (C
20:1
)
281.
2 (C
18:1
)
Inte
nsity
m/z
255.
2 (C
16:0
)PE 20:1p/16:0
724 726 728 730 732 7340
40
80
120
Inte
nsity
728.56
Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup
Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)
No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.
C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171
MALDI MS/MS: in-situ identification
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 22
0 100 200 300 400 500 600 7000
4000
8000
12000
16000
464.
3 (M
-C18
:1+H
2O)
446.
3 (M
-C18
:1)
436.
3 (M
-C20
:1+H
2O)
418.
3 (M
-C20
:1)
79.0
(PO
3- )
97.0
(H2P
O4- )
140
(pho
spho
etha
nola
min
e)
309.
3 (C
20:1
)
281.
2 (C
18:1
)
Inte
nsity
m/z
255.
2 (C
16:0
)
PE 20:1p/16:0
724 726 728 730 732 7340
40
80
120
Inte
nsity
728.56
Databases like Lipidmaps suggest only plasmalogen phospholipids with this mass and a phosphoethanolamine headgroup
Brain is rich in plasmalogen phospholipids (1 acyl link and 1 alkenyl link)
No neutral loss of the fatty acid linked by the alkenyl function in MS/MS analysis.
3 different isobaric lipid species, all from the same family, and all colocalized
PE 16:0p/20:1PE 18:0p/18:1
C. D. Cerruti, F. Benabdellah, O. Laprévote,D. Touboul, A. Brunelle, Anal. Chem. 2012, 84, 2164-2171
Non-alcoholic fatty liver disease(NAFLD)
Accumulation of lipids (mainly diacyl- and triacylglycerols) in hepatocytes,
The predominant risk factor for NAFLD appears to be insulin-resistance related to the metabolicsyndrome,
On stained sections, lipid vesicles are observed in fatty liver but are not observable in healthyliver,
NAFLD represents, in the absence of alcohol abuse, a wide spectrum of conditions ranging fromsimple steatosis (NAFL) to non-alcoholic steatohepatitis (NASH), that may progress to cirrhosisand end-stage liver disease.
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 23
1000 µm 1000 µm
Control liver Fatty liver
H&E staining (adjacent sections)
300 400 500 600 700 800 9000
1
2
3
4
5
6
Inte
nsity
per
uni
t are
a (c
ount
s x1
03 )
m/z
PC
DAG
Vit.E
Chol.
385.
33
369.34
339.
2931
3.28
523.47603.44
577.43551.43
430.36
758.55760.56
MAG
300 400 500 600 700 800 9000
5
10
15
20
Inte
nsity
per
uni
t are
a (c
ount
s x1
03 )
m/z
603.51
577.53
551.47
523.44
369.33
339.
28313.
27
Chol.
DAG
700 750 800 850 900 9500.0
0.1
0.2
0.3
0.4
0.5
853.69855.73857.74
758.45760.49
PC TAGMAG
Fatty liver - steatotic area
Control liver
Lipid profile of a fatty liver :Steatotic area, positive ion mode
Increase of the diacylglycerol signal in the steatotic area
Strong decrease of vitamin E signal Detection of triacylglycerols
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 24
TAG
DAG
MAG
Vit.E
D. Debois, M.P. Bralet, F. Le Naour, A. Brunelle, O. Laprévote, Anal. Chem. 2009, 81, 2823-2831
O
O
O
OP
O
OO
O
HOHO
HOOH
OH
X
X = H, Na, K, Ca...
O
HO
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 25
Fatty acids: O
OH
Sterols: Phospholipids:
α-TocopherolPhosphatidylcholine
Phosphatidylinositol
O
O
O
OP
O
O
O
ON
O
HN
OH
OP
O
OO
N
Sphingomyelin
HO Cholesterol
O
O
O
O
O O
Diglycerides: O
O
O
O
OH
Triglycerides:
Only mass spectrometry imaging makes possible to distinguish between the differentlocalizations among the same lipid species
Lipid profile of a fatty liver :Steatotic area, positive ion mode
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 26
100 µm
Fragments of TAGsFragments of TAGs and of other lipid species
In agreement with the diseasedescription
Cholesterol
16
0
179
0
7
0Sum of TAGs Sum of DAGs
19
0
50
0
79
0
64
0C30 DAGs C32 DAGs C34 DAGs C36 DAGs
D. Debois, M.P. Bralet, F. Le Naour, A. Brunelle, O. Laprévote, Anal. Chem. 2009, 81, 2823-2831
Lipid profile of a fatty liver :Steatotic area, correlation between DAGs and FAs
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 27
19
0
50
0
79
0
64
0C30 DAGs C32 DAGs C34 DAGs C36 DAGs
17
0
67
0
39
0
41
0
36
0Myristic acid C14:0 Sum of C16 FAs Stearic acid C18:0 Oleic acid C18:1 Linoleic acid C18:2
D. Debois, M.P. Bralet, F. Le Naour, A. Brunelle, O. Laprévote, Anal. Chem. 2009, 81, 2823-2831
Lipid profile of a fatty liver :DAGs C36
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 28
64
0C36 DAGs9
0
20
0
38
0
33
0
17
0C36:0 DAGsC36:1DAGsC36:2 DAGsC36:3 DAGsC36:4 DAGs
Composition of steatotic vesicles: Enrichment in cholesterol,
Lipids with longer acyl chains,
Enrichment in unsaturated lipids
D. Debois, M.P. Bralet, F. Le Naour, A. Brunelle, O. Laprévote, Anal. Chem. 2009, 81, 2823-2831
Chronic glaucoma
Second cause of blindness in the world
Due to a trabecular failure
Induces an increase of the intra-ocular pressure (IOP) and a progressive optic neuropathy
Symptoms are insidious, only a progressive visual loss
Early diagnostic can be made only by systematic IOP measurements during ophthalmologic examination
Glaucoma treatments Eye drops like prostaglandins derivatives or beta
blockers predominantly used as first line therapy to reduce the IOP (800 000 people in France in 2004) Addition of a preservative is needed, usually benzalkonium chloride (BAK)
Laser Surgical resection of the trabecular
Many studies have suspected BAK to induce a long-term toxicity in eye
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 29
Reduced visual field of view
Benzalkonium chloride
Quaternary ammonium salt with variable length alkyl chains, two predominant forms C12 and C14
Surfactant, antiviral and antifungal depending on the alkyl chain length
Toxic preservative: Damage of tissues due to oxidative stress Apoptosis and necrosis depending on the
concentration used and the time of application
Dry eye, burning and/or tingling sensation
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 30
R=C8 to C18 but mainly C12 and C14
C12
C14
Very easy to analyze by MALDI-MS or SIMS
MALDI-ToF/ToF imaging of whole rabbit eye sections
BAK detected in: cornea, conjunctiva, iridocorneal angle AND in and near the optic nerve
Confirmation of results with a round-robin experiment in 2 laboratories:(second data not shown)
Matrix deposition: TM-Sprayer (HTX Imaging) SunCollect (SunChrome)
MALDI spectrometers: 4800 MALDI TOF/TOF (AB-Sciex) Autoflex Speed (Bruker Daltonics)
Processing software: TissueView (AB Sciex) FlexImaging (Bruker Daltonics)
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 31
2 near to the optic nerve
3 optic nerve
1 cornea
Optical
1
2
3
1
2
3
TOF-SIMS imaging: small areas of rabbit eye sections
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 32
Each image:m/z 332.36 (BAK C14)
mc: 28500 µm x 500 µm256 x 256 pixelsPixel size 2 µm
3.1011 ions.cm-2 (Bi3+)
Limbus
Fornix
Conjunctiva
Limbus
Fornix
Conjunctiva
TrabeculumTrabeculum
Acquisition of 5 x 4 adjacent images in the iridocorneal angle(total area 2.5 mm x 2.0 mm)
Optical view (x 1.25)
Iridocorneal angle
Anatomy of a human eye
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 33
TOF-SIMS imaging: small areas of rabbit eye sections
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 34
Each image:m/z 332.36 (BAK C14)
mc: 28500 µm x 500 µm256 x 256 pixelsPixel size 2 µm
3.1011 ions.cm-2 (Bi3+)
Limbus
Fornix
Conjunctiva
Limbus
Fornix
Conjunctiva
TrabeculumTrabeculum
Acquisition of 5 x 4 adjacent images in the iridocorneal angle(total area 2.5 mm x 2.0 mm)
Optical view (x 1.25)
TOF-SIMS imaging: small areas of rabbit eye sections
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 35
1. F
orni
x
TUNEL:Fluorescence methodshowing apoptotic cells
2. T
rabe
culu
m
Optical
3. C
orne
a
m/z 332.36 (BAK C14) mc : 57
m/z 304.32 (BAK C12) mc : 60
m/z 304.32 (BAK C12) Redm/z 332.36 (BAK C14) Green
out
in
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE
Conclusion
TOF-SIMS and MALDI-TOF lipid imaging:
TOF-SIMS: micrometer resolution, no tissue preparation needed.
MALDI-TOF: in situ identifications of lipids.
For TOF-SIMS, many ions can also be evidenced by their co-localization with specific
fragments.
For both methods, the targeted compounds must be highly concentrated in well-defined
areas to be imaged.
Perspectives: data analysis, MS/MS, increase of TOF-SIMS sensitivity, neurodegenerative
diseases, liver diseases
36
1er Workshop EQUIPEX ANDROMEDE 12.06.2012 Alain BRUNELLE ICSN GIF SUR YVETTE 37
Agence Nationale de la Recherche Scientifique (ANR)
European Union(FP6 STREP Program and COST)
Institut de Chimiedes Substances Naturelles
Centre National de la Recherche Scientifique