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Chemical Imaging on Liver Steatosis
Using Synchrotron
Infrared and ToF-SIMS
Microspectroscopies
113/11/2012
Stéphane Nguyen
Plan
• Introduction
• Materials and Methods
• Results
• Discussion
213/11/2012
Introduction
• Steatosis: alcoholism, drug intake, small-bowel by-pass surgery or
metabolic syndrome
• Non alcoholic fatty...
Introduction
13/11/2012 4
DAG accretion
• Imbalance in the
uptake, synthesis, export
and oxidation of fatty acids
Formatio...
Problems
13/11/2012 5
Primary metabolic abnormalities leading to lipid
accretion
Local lipid composition
Materials and Methods:
Patients and liver samples
613/11/2012
doi:10.1371/journal.pone.0007408.t001
• Tissues fixed in for...
Materials and Methods:
Tissue section
713/11/2012
doi:10.1371/journal.pone.0007408.g001
PT: portal tract, BD: biliary
duct...
Materials and Methods:
Synchrotron FTIR microspectroscopy
• chemical composition in small sample area
• analysis at the di...
Materials and Methods:
ToF-SIMS Imaging
913/11/2012
• Ions are identified according to their mass-to-charge (m/z) ratio
• ...
Results:
Chemical imaging on steatosis using
FTIR microspectroscopy
1013/11/2012
doi:10.1371/journal.pone.0007408.g002
 O...
1113/11/2012
doi:10.1371/journal.pone.0007408.g003
Second derivatives of IR spectra
• slight downward shift of the CH2
sym...
1213/11/2012
doi:10.1371/journal.pone.0007408.g006
doi:10.1371/journal.pone.0007408.g007
• normal liver• non-steatotic
are...
Results:
Chemical imaging on steatosis using ToF-SIMS
mass spectrometry
1313/11/2012
doi:10.1371/journal.pone.0007408.g004...
1413/11/2012
doi:10.1371/journal.pone.0007408.g005
• DAG C30, C32, C34 and C36 bearing
saturated alkyl chains: outside of
...
Discussion
• synchrotron source to study the complete frequency range
from 900 to 4000 cm-1 (lipids)
• ToF-SIMS for invest...
Perspectives
• infrared spectroscopy might be used as a diagnosis mean
especially in the setting of liver transplantation
...
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Chemical Imaging on Liver Steatosis

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Chemical Imaging on Liver Steatosis Using Synchrotron
Infrared and ToF-SIMS Microspectroscopies

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Chemical Imaging on Liver Steatosis

  1. 1. Chemical Imaging on Liver Steatosis Using Synchrotron Infrared and ToF-SIMS Microspectroscopies 113/11/2012 Stéphane Nguyen
  2. 2. Plan • Introduction • Materials and Methods • Results • Discussion 213/11/2012
  3. 3. Introduction • Steatosis: alcoholism, drug intake, small-bowel by-pass surgery or metabolic syndrome • Non alcoholic fatty liver disease (obesity, insulin resistance, diabetes, drugs and the metabolic syndrome): most common cause of chronic liver disease in Western countries • precursor for steatohepatitis: a condition that may progress to cirrhosis and in some cases to the development of primary liver cancer • In countries with obesity: 25% of the general population and very common in overweight persons over the age of 30* 313/11/2012 *http://www.virtualmedicalcentre.com/diseases/fatty-liver-steatosis-steatohepatitis/648
  4. 4. Introduction 13/11/2012 4 DAG accretion • Imbalance in the uptake, synthesis, export and oxidation of fatty acids Formation of steatotic vesicles in the hepatocytes
  5. 5. Problems 13/11/2012 5 Primary metabolic abnormalities leading to lipid accretion Local lipid composition
  6. 6. Materials and Methods: Patients and liver samples 613/11/2012 doi:10.1371/journal.pone.0007408.t001 • Tissues fixed in formalin • One specimen of non-tumorous liver snap frozen in liquid nitrogen and stored at -80°C
  7. 7. Materials and Methods: Tissue section 713/11/2012 doi:10.1371/journal.pone.0007408.g001 PT: portal tract, BD: biliary duct, PV: portal vein, HA: hepatic artery, CLV: centrilobular vein, SV: steatotic vacuole. • Normal hepatic lobule • fatty liver area : exhibiting macrovacuolar and microvesicular steatosis X100 X400
  8. 8. Materials and Methods: Synchrotron FTIR microspectroscopy • chemical composition in small sample area • analysis at the diffraction while preserving a high spectral quality 813/11/2012 http://www.chemicool.com/definition/fourier_transform_infrared_spectrometer_ftir.html
  9. 9. Materials and Methods: ToF-SIMS Imaging 913/11/2012 • Ions are identified according to their mass-to-charge (m/z) ratio • Accessible mass range about 1500 Da • Ensured a spatial resolution of 1–2 mm and a mass resolution M/DM=104 http://www.geomuseum.uni-goettingen.de:8001/people/vthiel/tof_sims/index.shtml
  10. 10. Results: Chemical imaging on steatosis using FTIR microspectroscopy 1013/11/2012 doi:10.1371/journal.pone.0007408.g002  Optical image of steatotic hepatocytes Steatotic hepatocytes  Chemical imaging of some bands Non-steatotic hepatocytes
  11. 11. 1113/11/2012 doi:10.1371/journal.pone.0007408.g003 Second derivatives of IR spectra • slight downward shift of the CH2 symmetric and anti-symmetric stretching modes in steatosis vesicles • lipids in a different environment, probably with higher structural order Results: Chemical imaging on steatosis using FTIR microspectroscopy
  12. 12. 1213/11/2012 doi:10.1371/journal.pone.0007408.g006 doi:10.1371/journal.pone.0007408.g007 • normal liver• non-steatotic area of fatty liver Biopsies stained with HES (hematoxylin, eosin and safran) (x400) FTIR spectroscopy on periportal hepatocytes • higher intensity of CH3 and CH2 bands (2800–3000 cm-1) and esters (1710–1760 cm-1) • Changes in the frequency domains 950–1200 cm-1 corresponding in part to sugar contribution • small lipid droplets exhibiting sizes less than 10 mm containing DAGs C=O CH2-CH3 Results: Spectroscopic analysis of non-steatotic hepatocytes in fatty liver
  13. 13. Results: Chemical imaging on steatosis using ToF-SIMS mass spectrometry 1313/11/2012 doi:10.1371/journal.pone.0007408.g004 doi:10.1371/journal.pone.0007408.g005 Positive ion mode video • DAG C36 inside • Overlay: DAG C36 and cholesterol co-localized in steatotic vesicles MAG cholesterol • DAG C30 outside of steatotic vesicles
  14. 14. 1413/11/2012 doi:10.1371/journal.pone.0007408.g005 • DAG C30, C32, C34 and C36 bearing saturated alkyl chains: outside of steatotic vesicles • DAG containing at least one unsaturated acyl chain: concentrated into steatotic droplets Results: Chemical imaging on steatosis using ToF-SIMS mass spectrometry
  15. 15. Discussion • synchrotron source to study the complete frequency range from 900 to 4000 cm-1 (lipids) • ToF-SIMS for investigating the local composition and distribution of the molecular species of lipids • investigation at cellular and subcellular levels • Concentration of unsaturated lipids inside steatotic vesicles may constitute a potential highly reactive site for peroxidation 1513/11/2012
  16. 16. Perspectives • infrared spectroscopy might be used as a diagnosis mean especially in the setting of liver transplantation • spatial resolution and sensitivity of synchrotron FTIR microspectroscopy and mass spectrometry may open new avenue for characterizing early events in pathologies or for identifying markers for diagnosis and prognosis • FTIR microspectroscopy using conventional infrared source might be set up in hospitals for clinical use 1613/11/2012

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