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

1. Chemical Imaging on Liver Steatosis
Using Synchrotron
Infrared and ToF-SIMS
Microspectroscopies
113/11/2012
Stéphane Nguyen

2. Plan
• Introduction
• Materials and Methods
• Results
• Discussion
213/11/2012

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. Introduction
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DAG accretion
• Imbalance in the
uptake, synthesis, export
and oxidation of fatty acids
Formation of steatotic
vesicles in the
hepatocytes

5. Problems
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Primary metabolic abnormalities leading to lipid
accretion
Local lipid composition

6. Materials and Methods:
Patients and liver samples
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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. Materials and Methods:
Tissue section
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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. Materials and Methods:
Synchrotron FTIR microspectroscopy
• chemical composition in small sample area
• analysis at the diffraction while preserving a high spectral
quality
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http://www.chemicool.com/definition/fourier_transform_infrared_spectrometer_ftir.html

9. Materials and Methods:
ToF-SIMS Imaging
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• 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. Results:
Chemical imaging on steatosis using
FTIR microspectroscopy
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doi:10.1371/journal.pone.0007408.g002
 Optical image of
steatotic
hepatocytes
Steatotic
hepatocytes
 Chemical imaging of
some bands
Non-steatotic
hepatocytes

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. 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. Results:
Chemical imaging on steatosis using ToF-SIMS
mass spectrometry
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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

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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. 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
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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
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