Article pubs.acs.org/ac
In Situ Chemical Composition Analysis of Cirrhosis by Combining Synchrotron Fourier Transform Infrared and Synchrotron X‑ray Fluorescence Microspectroscopies on the Same Tissue Section François Le Naour,*,†,‡ Christophe Sandt,§ Chengyuan Peng,†,‡ Nicolas Trcera,§ Franck Chiappini,†,‡ Anne-Marie Flank,§ Catherine Guettier,†,‡,⊥ and Paul Dumas§ †
INSERM, Unité 785, Avenue Paul Vaillant Couturier, Villejuif F-94800, France Université Paris-Sud, UMR-S 785, Villejuif F-94800, France § Synchrotron SOLEIL, Gif-sur-Yvette F-91192, France ⊥ Service d’Anatomie Pathologique, AP-HP Hôpital Paul Brousse, Villejuif F-94800, France ‡
S Supporting Information *
ABSTRACT: Liver is subject to various chronic pathologies, progressively leading to cirrhosis, which is associated with an increased risk of hepatocellular carcinoma. There is an urgent need for diagnostic and prognostic markers of chronic liver diseases and liver cancer. Spectroscopy-based approaches can provide an overview of the chemical composition of a tissue sample offering the possibility of investigating in depth the subtle chemical changes associated with pathological states. In this study, we have addressed the composition of cirrhotic liver tissue by combining synchrotron Fourier transform infrared (FTIR) microspectroscopy and synchrotron micro-X-ray fluorescence (XRF) on the same tissue section using a single sample holder in copper. This allowed investigation of the in situ biochemical as well as elemental composition of cells and tissues at high spatial resolution. Cirrhosis is characterized by regeneration nodules surrounded by annular fibrosis. Hepatocytes within cirrhotic nodules were characterized by high content in esters and sugars as well as in phosphorus and iron compared with fibrotic septa. A high heterogeneity was observed between cirrhotic nodules in their content in sugars and iron. On fibrosis, synchrotron XRF revealed enrichment in calcium compared to cirrhotic hepatocytes. Careful scrutiny of tissue sections led to detection of the presence of microcrystals that were demonstrated as precipitates of calcite using synchrotron FTIR. These results demonstrated that synchrotron FTIR and synchrotron XRF microspectroscopies provide complementary information on the chemical composition of cirrhotic hepatocytes and fibrotic septa in cirrhosis.
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nucleic acids, carbohydrates, and lipids, all of which have specific absorption bands in the IR frequency domain. Thus, IR spectroscopy is a very valuable tool for biochemical investigations. Fourier transform infrared (FTIR) microspectroscopy combines IR spectroscopy and microscopy for determining the chemical composition in a small sample area. The application of synchrotron radiation as a high-brightness source of IR photons has brought the technique to achieving analysis at the diffraction limit (typically, half the wavelength of the vibrational frequency) while preserving a high spectral quality.5−10 We have investigated previously the chemical composition of steatotic vesicles in fatty liver using FTIR microspectroscopy and demonstrated its ability to discriminate early stages of steatosis with normal liver.11 On the other hand, the elemental composition of a biological tissue can be addressed by using X-ray fluorescence (XRF). This method is
iver is subject to chronic diseases that can be induced by different factors such as dysmetabolic syndrome, alcohol, or viral hepatitis (hepatitis B or C virus). Under these different conditions, chronic hepatocellular injury characterized by necrosis and inflammation generates fibrogenesis, which culminates in liver cirrhosis. Continuous cycles of this destructive−regenerative process foster liver carcinogenesis. Indeed, cirrhosis constitutes the most important risk factor of hepatocellular carcinoma (HCC), which is a leading cause of death from cancer worldwide.1−4 Dramatic changes in the biochemical and elemental composition of the liver tissue occur during chronic liver diseases. Knowledge about these changes may provide new insights into the understanding of mechanisms underlying these pathologies and would help to characterize diagnostic and prognostic markers. Spectroscopy-based approaches are potentially useful in addressing the chemical composition and distribution of components across a biological tissue sample. IR spectroscopy is based on the determination of absorption of IR light due to resonance with vibrational motions of functional molecular groups. Biological tissue is essentially made up of proteins, © 2012 American Chemical Society
Received: July 22, 2012 Accepted: November 2, 2012 Published: November 3, 2012 10260
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normal livers (not shown) were also provided by CRB ParisSud. Access to this material was in agreement with French ethical laws. Tissues were fixed in formalin for routine pathological assessment, and one fragment was immediately snap-frozen in liquid nitrogen and stored at −80 °C until use. Serial sections were cut from frozen specimens with 6 μm thickness at −20 °C with a CM3050-S cryostat (Leica Microsystèmes SAS, France) and alternately deposited on a glass slide for histological control or on a copper sample holder for both FTIR and XRF microspectroscopies. Sections for histology were stained with hematoxylin, eosin, and safran (HES). Sections for microspectroscopy were dried at room temperature. FTIR Microspectroscopy. Synchrotron IR microspectroscopy was performed at the SMIS beamline at the SOLEIL Synchrotron Radiation Facility (Saint-Aubin, France) operating at 2.75 GeV with a current of 400 mA delivered in top-up mode. Details of the experimental procedure have already been described.11,23 IR photons are created by the electrons deflected from a bending magnet in the storage ring.24 The IR photon source is coupled to a Thermo Fischer NEXUS Nicolet 5700 FTIR spectrometer. Attached to the spectrometer is a CONTINUUM XL microscope (Thermo Scientific, CA). The detector of the IR microscope is a liquid-nitrogen-cooled mercury−cadmium−telluride (MCT-A) detector (50 μm). The microscope operated in confocal mode, using a 32× infinitycorrected Schwarzschild objective (NA = 0.65) and a matching 32× condenser. All spectra were obtained using a double-path single-masking aperture (confocal arrangement) size set to 10 μm × 10 μm. The spectra were collected in the 4000−800 cm−1 mid-IR range at a resolution of 8 cm−1 with 16 coadded scans. Each spectrum was recorded in approximately 10 s. IR microspectroscopy was also performed on an IN10MX microscope (Thermo Scientific) for recording large maps. All spectra were collected by an ultrafast mode using a 50 μm × 50 μm aperture. The spectra were collected in the 4000−800 cm−1 mid-IR range at a resolution of 16 cm−1 with one spectrum per pixel. Data analysis of IR spectra and chemical images was performed using OMNIC software (Thermo Scientific). XRF. The synchrotron XRF experiments have been carried out on the LUCIA beamline25 at the SOLEIL synchrotron. The sample is excited with a monochromatic incident X-ray beam of 7.3 keV (above the Fe K-edge) provided by the Si(111) doublecrystal monochromator. The beam is microfocused thanks to two dynamically bendable mirrors in the Kirkpatrick-Baez (KB) configuration. A sample holder in high-purity copper was used. Copper, with its excitation K-edge at 8980 eV, is the best element for the LUCIA beamline. The cross section of the beam was 3 μm × 3 μm at the position of the sample. The sample is raster-scanned, and in each point, the full XRF spectrum is recorded using an energy-dispersive detector (silicon drift diode) with an energy resolution of ∼175 eV. Synchrotron XRF maps were obtained using Igor Pro 6 software. To collect the elemental maps, we selected various regions of interest from the fluorescence spectra. The maps for the calcium were collected by taking into account a very narrow region between 3640 and 3780 eV corresponding to the Ca Ka line and the tail of the K Kb line. Using this configuration, elemental maps have been collected with various sizes. Large maps of 6 mm (H) × 5 mm (V) have been collected with a step of 50 μm, whereas we used a step of 15 μm for small maps of 500 μm (H) × 500 μm (V). In both cases, the counting time used to record the fluorescence spectra was equal to 4 s.
based on the detection of X-rays emitted from sample atoms excited with X-rays of high energy. XRF analysis is multielemental, highly sensitive, and quantitative because the intensity of fluorescence is directly proportional to the concentration of the element within the sample. Over the past decade, the investigation of biological samples has been favored by the developments of high-flux X-ray and highfocused beam.12−17 XRF analysis allowed investigation of the hepatic iron distribution18 as well as the distribution of metalloproteins in HCC and surrounding tissues.19 Finally, the combination of FTIR and XRF allows the investigation of both the biochemical and elemental compositions on the same sample. However, only a few studies have been reported using such a combination,20−22 and none of them was performed on liver tissue. In this report, we addressed the possibility of coupling FTIR and synchrotron XRF microspectroscopies on the same tissue section deposited on a copper sample holder. The combination of these two microspectroscopies leads to a comprehensive characterization of the composition of liver cirrhotic tissue.
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MATERIALS AND METHODS Samples and Tissue Sections. Frozen tissue specimens from the native liver of three patients transplanted for alcoholic cirrhosis and of three normal livers (Table 1) were obtained Table 1. Patients and Origin of Surgical Liver Samples Investigated with FTIR and XRF age
pathological diagnosis
F
50
normal liver
2
F
38
normal liver
3
F
30
normal liver
4
M
62
5
M
41
6
M
67
alcoholic cirrhosis alcoholic cirrhosis alcoholic cirrhosis
patient
sex
1
associated diagnosis liver metastasis from colorectal cancer focal nodular hyperplasia focal nodular hyperplasia low-grade dysplastic nodules2 none macroregenerative nodule1
iron load on Perls staining no iron overload no iron overload no iron overload slight iron overload no iron overload slight iron overload
from the Centre de Ressources Biologiques (CRB) Paris-Sud. A total of 7 additional cirrhosis livers (Table 2) and 11 additional Table 2. Patients and Origin of Surgical Liver Samples Investigated for the Presence of Calcite patient
sex
age
7
M
66
8
M
66
9
M
67
10
M
75
11
M
59
12
M
65
13
F
51
pathological diagnosis
associated diagnosis
iron load on Perls staining
crystals of calcite
alcoholic cirrhosis alcoholic cirrhosis alcoholic cirrhosis HBV cirrhosis HBV/HIV cirrhosis HCV cirrhosis HCV/HIV cirrhosis
HCC
slight iron overload slight iron overload moderate iron overload moderate iron overload moderate iron overload moderate iron overload no iron overload
−
HCC HCC HCC HCC HCC diffuse HCC
+ + − + − −
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performed on a restricted frequency domain (1450−1800 cm−1). In our hand, we have observed that Ultralene leads to low-quality spectra in the energy domain 800−1400 cm−1. In addition, this film is very flexible and thus not convenient for manipulating thin tissue sections. Therefore, a sample holder allowing XRF analysis compatible with investigations in the mid-IR frequency domain is still lacking. Thus, we checked the possibility of realizing FTIR analysis on a copper sample holder that is reflecting IR light as a mirror and a support compatible with XRF analysis below the K-edge of copper at 8980 eV. Tissue sections from frozen specimens of liver cirrhosis were deposited on the copper sample holder followed by FTIR acquisition using an internal light source or synchrotron radiation. IR and fluorescence spectra were collected on two POIs (POI 1 and POI 2), which were located respectively in the cirrhotic nodule and fibrous septa (Figure 2A). FTIR microscopy led to visualization of the distribution of lipids (2800−3000 cm−1), esters (1710−1780 cm−1), proteins (1475−1710 cm−1), collagen (1190−1350 cm−1), and sugars (900−1190 cm−1) with very good sensitivity and signal-to-noise ratio on the copper (Figure 2B). In addition, synchrotron XRF acquisition on the same POI leads to detection of the presence of the elements phosphorus, sulfur, chlorine, potassium, calcium, and iron (Figure 2C). The possibility of investigating the biochemical and elemental compositions on the same tissue section with a high spatial resolution was then feasible. Furthermore, superimposition of the IR or XRF spectra of the two POIs allowed observation of the different level intensities of lipids, esters, collagen, and sugars as well as of elements such as phosphorus, calcium, and iron (Figure 2B,C). These observations suggested different biochemical and elemental compositions between cirrhotic hepatocytes and fibrosis. The cirrhotic liver was further investigated by chemical imaging using both microspectroscopies. The chemical imaging on the whole tissue section using FTIR microspectroscopy at low spatial resolution (50 μm × 50 μm) allowed visualization of the distribution of lipids, esters, proteins, collagen, and sugars (Figures 3A and S1 in the Supporting Information). Proteins were observed to be mostly enriched in fibrosis areas, in which collagen exhibited a selective enrichment. By contrast, the distribution of lipids including ester lipid species and sugars showed a selective enrichment inside the cirrhotic nodule. Furthermore, chemical imaging on the whole tissue section provided an overview of the heterogeneity of cirrhotic nodules. In particular, high heterogeneity in sugars was observed in some nodules, whereas the lipid content was quite homogeneous. It has been previously reported that liver cirrhosis is accompanied by quantitative and qualitative changes in glycogen.28,29 One possibility that could account for the observed heterogeneous sugar content is loss of the metabolic zonation present in the normal liver due to the formation of regenerative nodules in cirrhosis. In the process of liver lesion, some cirrhotic nodules could partially preserve the initial structure and metabolic zonation. However, even in this case, essential changes occur in the composition and probably the metabolism of sugars.29,30 Cirrhotic tissue was further investigated using synchrotron XRF. Elemental maps for phosphorus, calcium, and iron were collected (Figure 3A). The large maps allowed visualization of the important differences in the distribution of the various elements in the sample. The distribution of phosphorus was observed to be highly enriched into the cirrhotic nodules. The distribution of phosphorus is in accordance with that of lipids
Fluorescence spectra for points of interest (POI) in the cirrhotic nodules and in the fibrous septa were also collected with a counting time of 120 s for each point.
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RESULTS AND DISCUSSION Cirrhosis is the end stage of chronic liver disease. It is characterized morphologically by regeneration nodules surrounded by annular fibrosis (Figure 1). In order to investigate
Figure 1. Histological features of normal liver and cirrhosis. Tissue sections of 4 μm thickness were taken from normal liver or from cirrhosis and stained with HES. Normal hepatic lobule or cirrhosis exhibiting nodules surrounded by fibrosis are shown (upper panel, 25×; lower panel, 100×). Abbreviations: PT, portal tract; BD, biliary duct; PV, portal vein; HA, hepatic artery; CLV, centrilobular vein; N, nodule; F, fibrosis.
the global biochemical composition as well as the elemental composition of cirrhosis, we have addressed the possibility of combining synchrotron FTIR and XRF on the same tissue section. Only a few reports have addressed the combination of different types of spectroscopies.20,23,26,27 The coupling of several spectroscopy-based approaches on the same tissue section is highly dependent on the possibility of performing the acquisitions on a single sample holder. The combination on the same tissue section of synchrotron FTIR with synchrotron UV as well as mass spectrometry has been demonstrated by using a gold-coated glass slide.23 Moreover, this type of sample holder does not allow one to perform XRF because of the presence of traces of element in glass and because M lines of the gold are around 2−3 keV in the region of the P, S, and Cl K lines. A new sample substrate has been reported for imaging and correlating organic and trace metal compositions in biological cells and tissues.27 Thus, a gold grid was developed for combining synchrotron FTIR and XRF. However, the XRF study was focused on iron, copper, and zinc in the energy domain 5−10 keV, and FTIR analysis was performed in a restricted frequency domain (1200−1800 cm−1). Finally, the combination of synchrotron FTIR and XRF has been performed using an Ultralene film.20 Such a sample holder allowed XRF analysis in the energy domain 0−10 keV, but FTIR investigation was again 10262
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Figure 2. FTIR and XRF on a copper sample holder. (A) HES staining on cirrhotic tissue. Synchrotron FTIR and XRF acquisitions were performed on serial tissue sections on two POIs corresponding to cirrhotic hepatocytes (POI 1) or fibrosis (POI 2). The black square is of 10 μm × 10 μm size. The FTIR (B) or XRF (C) spectra are shown.
and sugars, which are biochemical components exhibiting high content in phosphorus (e.g., phosphatidyl choline or
phosphatidyl serine in the composition of biological membranes). The distribution of iron allowed also the observation 10263
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Figure 3. Chemical imaging on liver cirrhosis using FTIR and synchrotron XRF microspectroscopies. (A) Chemical imaging of lipids, proteins, and sugars was investigated on the whole tissue section of cirrhosis using an IR internal source with 50 μm × 50 μm aperture size. The distribution of iron, calcium, and phosphorus was investigated on the same whole tissue section of liver cirrhosis by synchrotron XRF with a spatial resolution 50 μm × 50 μm. The close-up section labeled with a window corresponds to a cirrhotic nodule on which acquisitions were performed (B) using both synchrotron FTIR experiments with 10 μm × 10 μm aperture size and synchrotron XRF with the spatial resolution 15 μm × 15 μm.
The high content of calcium observed in fibrosis from three patients was confirmed in seven additional patients with cirrhosis, suggesting that the calcium content is a general feature of fibrosis. This high level of calcium raises the question of a possible mineralization. Additional tissue sections were performed for a careful scrutiny of the presence of microcrystals. We took advantage of the possibility to investigate the composition of such microcrystals by FTIR using the high spatial resolution of synchrotron radiation.31 For each patient, a frozen tissue section was performed, on which up to 5 maps (200 μm × 200 μm) were investigated. IR spectra exhibited spectral features, suggesting the presence of calcite (Figure 4A). The distribution of some bands corresponding to proteins, collagen, or specific peaks of calcite was visualized on the maps (Figure 4B). This demonstrated the selective distribution of the peaks of calcite on the microcrystals. The presence of crystals of calcite was observed in three out of seven patients (Table 2). It should be noted that samples from 12 normal livers were investigated under the same experimental conditions and no microcrystal was found (data not shown). Altogether, these results suggest that a high level of calcium may be accompanied by the mineral calcification of cirrhosis. The important increase in the calcium content observed in fibrosis has never been reported. Recently, calcification of hepatocytes has been reported in liver grafts after transplantation32−34 following severe ischemia-reperfusion injury. Ischemic stress has been
of enrichment in cirrhotic nodules. However, an overview on the whole tissue section indicates a high heterogeneity of the iron content between the cirrhotic nodules. Indeed, some nodules were poor in iron, whereas others were highly enriched. A comparison of the elemental compositions between the hepatocellular nodules and fibrosis allowed the observation of an important variation in the calcium content, which was anticorrelated with phosphorus and iron. The calcium-rich regions fitted very well with the distribution of proteins as well as collagen previously imaged using FTIR, thus corresponding to fibrosis areas. It should be noted that chemical imaging analysis was also performed on tissue sections from normal liver. The distribution of biochemical components and elements was quite homogeneous (data not shown). The study was further focused on a single cirrhotic nodule at high spatial resolution (10 μm × 10 μm) using synchrotron radiation. The distribution of sugars, esters, and lipids appeared to be heterogeneous (Figure 3B), suggesting a high variability in the biochemical content at the cellular level. The distribution of iron mainly in the nodule compared to the calcium distribution located in the fibrosis was confirmed (Figure 3B). It has been suggested that cirrhotic hepatocytes exhibit a heterogeneous composition.30 Here, we demonstrate that FTIR and XRF microspectroscopies open new avenues for investigating the chemical heterogeneity of cirrhotic hepatocytes at the cellular level. 10264
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Figure 4. Presence of calcite in fibrosis. (A) A microcrystal was observed in fibrosis. Synchrotron FTIR spectra were acquired on fibrosis (blue cross and blue spectrum) or on the microcrystal (red cross and red spectrum). The FTIR spectrum corresponding to pure calcite is shown in black. Peaks corresponding to the spectral signature of calcite are labeled with black arrows. (B) The distribution of proteins and collagen as well as of peaks of calcite is shown. Scale bar: 10 μm.
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previously shown to induce calcium accumulation at the cell level, by either impaired oxidative metabolism or abnormalities in the plasma membrane. This elevated intracellular calcium concentration is responsible for cytoskeleton modifications, which alter the cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally mitochondrial calcification. In the calcified areas, myofibroblasts expressing bone-specific matrix proteins such as osteopontin and type 1 collagen were abundant. These observations suggested that liver calcification following transplantation may be a consequence of precipitation of calcium phosphate, likely hydroxyapatite emanating from necrotic or apoptotic hepatocytes associated with proliferation of myofibroblasts expressing bone-specific matrix proteins. Finally, hepatocellular calcification was associated with a poor graft outcome. Further investigations will be required to understand the mechanism and mode of calcification in cirrhosis. The detection of calcite crystals indicates rather an extracellular distribution of calcium, suggesting a different mechanism than that occurring in ischemic injury. The clinical relevance will also have to be investigated. Indeed, calcification could be a marker for the age of the fibrosis or for its remodelling ability. The presence of calcium and calcification in fibrosis could be responsible for the stiffness of cirrhotic liver that is related to poor prognosis.
CONCLUSIONS
This is the first report combining synchrotron FTIR and XRF microspectroscopies using a copper sample holder. Such a sample holder provides a good signal-to-noise ratio and thus high-quality spectra in the mid-IR frequency domain as well as in the 0−8 keV energy domain. This allows the investigation on the same tissue section of the in situ biochemical and elemental compositions at a high spatial resolution typically at the cellular level. In addition, major advantages of these two types of spectroscopies for investigating biological tissue are that they do not require any treatment, labeling, or staining of the sample and they are both compatible with tissue sections routinely performed at the hospital. Thus, a copper sample holder can be compatible with analysis of a large number of biological samples and could also be reused after washing. The high interest of a combination yielding complementary information on the chemical composition of cells and tissues was illustrated by the high content of calcium observed in fibrosis using synchrotron XRF, whereas the presence of microcrystals of calcite was demonstrated by synchrotron FTIR. In conclusion, the combination of FTIR and XRF microspectroscopies may provide a better understanding of the chemical changes occurring in pathologies and may open new avenues for the characterization of spectral and molecular markers. 10265
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(23) Petit, V. W.; Réfrégiers, M.; Guettier, C.; Jamme, F.; Sebanayakam, K.; Brunelle, A.; Laprévote, O.; Dumas, P.; Le Naour, F. Anal. Chem. 2010, 82, 3963−3968. (24) Dumas, P.; Polack, F.; Lagarde, B.; Chubar, O.; Giorgetta, J. L.; Lefrançois, S. Infrared Phys. Technol. 2006, 49, 152−160. (25) Flank, A.-M.; Cauchon, G.; Lagarde, P.; Bac, S.; Janousch, M.; Wetter, R.; Dubuisson, J.-M.; Idir, M.; Langlois, F.; Moreno, T.; Vantelon, D. Nucl. Instrum. Methods B 2006, 246, 269−274. (26) Briois, V.; Vantelon, D.; Villain, F.; Couzinet, B.; Flank, A.-M.; Lagarde, P. J. Synchrotron Radiat. 2007, 14, 403−408. (27) Miller, L. M.; Wang, Q.; Smith, R. J.; Zhong, H.; Elliott, D.; Warren, J. Anal. Bioanal. Chem. 2007, 387, 1705−1715. (28) Diem, M.; Chiriboga, L.; Yee, H. Biopolymers 2000, 57, 282− 290. (29) Kudryavtseva, M.; Bezborodkina, N. N.; Okovity, S. V.; Kudryavtsev, B. N. Eur. J. Gastroenterol. Hepatol. 2001, 13, 693−697. (30) Krähenbühl, L.; Lang, C.; Lüdes, S.; Seiler, C.; Schäfer, M.; Zimmermann, A.; Krähenbühl, S. Liver Int. 2003, 23, 101−109. (31) Dessombz, A.; Bazin, D.; Dumas, P.; Sandt, C.; Sule-Suso, J.; Daudon, M. PLoS One 2011, 6, e28007. (32) Tzimas, G. N.; Afshar, M.; Chevet, E.; Emadali, A.; Vali, H.; Metrakos, P. P. BMC Surg. 2004, 4, 9. (33) Kalantari, F.; Miao, D.; Emadali, A.; Tzimas, G. N.; Goltzman, D.; Vali, H.; Chevet, E.; Auguste, P. Mod. Pathol. 2007, 20, 357−366. (34) Talmon, G. A.; Wisecarver, J. L. Ultrastruct. Pathol. 2010, 34, 362−365.
ASSOCIATED CONTENT
S Supporting Information *
Chemical imaging on liver cirrhosis using FTIR microspectroscopy. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: (33) 1 45 59 60 77. Fax (33) 1 45 59 60 90. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by PRES UniverSud Paris, Fondation pour la Recherche Médicale, and Comité Ile de France of Ligue Nationale Contre le Cancer. It was also supported by beam time allocations at synchrotron SOLEIL (Proposals 20090493, 20100292, and 20110706). We are grateful to the SOLEIL staff for general facilities placed at our disposal. We are grateful to Mathieu Wavelet and Ibraheem Yousef for skillful technical assistance.
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