Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis (original) (raw)
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A robust collagen scoring method for human liver fibrosis by second harmonic microscopy
Optics Express, 2010
Second Harmonic Generation (SHG) microscopy offers the opportunity to image collagen of type I without staining. We recently showed that a simple scoring method, based on SHG images of histological human liver biopsies, correlates well with the Metavir assessment of fibrosis level (Gailhouste et al., J. Hepatol., 2010). In this article, we present a detailed study of this new scoring method with two different objective lenses. By using measurements of the objectives point spread functions and of the photomultiplier gain, and a simple model of the SHG intensity, we show that our scoring method, applied to human liver biopsies, is robust to the objective's numerical aperture (NA) for low NA, the choice of the reference sample and laser power, and the spatial sampling rate. The simplicity and robustness of our collagen scoring method may open new opportunities in the quantification of collagen content in different organs, which is of main importance in providing diagnostic information and evaluation of therapeutic efficiency. Stone, "Urinary assays for desmosine and hydroxylysylpyridinoline in the detection of cirrhosis,"
Scientific Reports, 2017
Polarization dependence second harmonic generation (P-SHG) microscopy is gaining increase popularity for in situ quantification of fibrillar protein architectures. In this report, we combine P-SHG microscopy, new linear least square (LLS) fitting and modeling to determine and convert the complex second-order non-linear optical anisotropy parameter ρ of several collagen rich tissues into a simple geometric organization of collagen fibrils. Modeling integrates a priori knowledge of polyhelical organization of collagen molecule polymers forming fibrils and bundles of fibrils as well as Poisson photonic shot noise of the detection system. The results, which accurately predict the known sub-microscopic hierarchical organization of collagen fibrils in several tissues, suggest that they can be subdivided into three classes according to their microscopic and macroscopic hierarchical organization of collagen fibrils. They also show, for the first time to our knowledge, intrahepatic spatial discrimination between genuine fibrotic and non-fibrotic vessels. CCl 4-treated livers are characterized by an increase in the percentage of fibrotic vessels and their remodeling involves peri-portal compaction and alignment of collagen fibrils that should contribute to portal hypertension. This integrated P-SHG image analysis method is a powerful tool that should open new avenue for the determination of pathophysiological and chemo-mechanical cues impacting collagen fibrils organization. Collagens play a central role in the formation of fibrils networks involved in the architecture of tissues and organs. In extracellular matrixes (ECM), the physical compressive and tensile strains generated by cell traction are key mechanisms involved in the long-range ordering and remodeling of collagen fibrils 1,2. These fibrils, consisting of long and filamentous polymers of collagen molecules, are arrays of axial and lateral supramolecular assembly of quarter-staggered collagen molecules resulting in 67 nm periodic striation observed at ultrastructural level with transmission electron microscopy. While much is known about the different hierarchical level of fibrillogenesis, comparatively little is known about how collagen fibrils assemble together into the diverse supramolecular arrangements found in the body. Moreover, remodeling of collagen fibrils involved in several pathologies encompassing fibrosis, cancer, bone and several connective tissues diseases also awaits a precise 3D description. Label-free second harmonic generation (SHG) process relies on a nonlinear optical interaction with hyperpolarizable non centrosymetric endogenous fibrillar proteins like collagen and myosin causing scattered coherent radiation at twice the fundamental frequency 3,4. Thus, it has proved to be an extremely beneficial contrast mechanism for label-free imaging of these endogenous molecules in situ, in vivo, in physiological as well as in disease state. Polarization dependence second harmonic generation (P-SHG) microscopy that enables quantification of
Scientific Reports, 2015
Liver fibrosis assessment by biopsy and conventional staining scores is based on histopathological criteria. Variations in sample preparation and the use of semi-quantitative histopathological methods commonly result in discrepancies between medical centers. Thus, minor changes in liver fibrosis might be overlooked in multi-center clinical trials, leading to statistically non-significant data. Here, we developed a computer-assisted, fully automated, staining-free method for hepatitis B-related liver fibrosis assessment. In total, 175 liver biopsies were divided into training (n = 105) and verification (n = 70) cohorts. Collagen was observed using second harmonic generation (SHG) microscopy without prior staining, and hepatocyte morphology was recorded using two-photon excitation fluorescence (TPEF) microscopy. The training cohort was utilized to establish a quantification algorithm. Eleven of 19 computer-recognizable SHG/TPEF microscopic morphological features were significantly correlated with the ISHAK fibrosis stages (P < 0.001). A biphasic scoring method was applied, combining support vector machine and multivariate generalized linear models to assess the early and late stages of fibrosis, respectively, based on these parameters. The verification cohort was used to verify the scoring method, and the area under the receiver operating characteristic curve was >0.82 for liver cirrhosis detection. Since no subjective gradings are needed, interobserver discrepancies could be avoided using this fully automated method. Hepatitis B is a major infectious disease in Asia. Patients with chronic hepatitis B carry high liver cirrhosis and liver cancer risks 1,2. Before the development of antiviral therapies, there was no efficient strategy for preventing chronic hepatitis B from progressing to end-stage liver disease 3,4. According to reports from the World Health Organization, 2 billion people are estimated to have experienced a hepatitis B virus (HBV) infection during their lifetime, and more than 350 million are chronically infected. In addition, approximately 600,000 people die annually from acute or chronic HBV infection sequelae. Liver fibrosis develops in patients with chronic hepatitis B because of repeated necroinflammation, followed by hepatocyte regeneration and the accumulation of extracellular matrix (ECM) proteins, such as collagen 5,6. When hepatocytes are injured, the immune system is activated, and growth factors are released that activate hepatic stellate cells to produce collagen and other fibrillar components 7. These substances are deposited into the ECM space. During liver injury, the collagen degradation process is impaired, which further leads to excessive ECM protein accumulation 8. The end stage of liver fibrosis is liver cirrhosis, which can lead to liver cancer or liver failure.
Accordion-like collagen fibrils suggested by P-SHG image modeling : implication in liver fibrosis
2018
ABSTRACTSecond-order non-linear optical anisotropy parameter ρ = χ33 / χ31 is calculated for collagen-richt issues considering both a single dominant molecular hyperpolarizability tensor element β333 = β at single helix level and a priori known submicrometric triple helical organization of collagen molecules. Modeling is further improved by taking account of Poisson photonic shot noise of the detection system and simple supra-molecular fibrillar arrangements in order to accurately simulate the dispersion of ρ values in collagen-rich tissues such as tendon, skin and liver vessels. From combined P-SHG experiments and modeling, we next correlate experimental and theoretical statistical distributions of ρ. Our results highlight that the dispersion of experimental ρ values is mainly due to (i) Poisson photonic shot noise in tendon and skin, which proves to have a preponderant effect in P-SHG experiments (ii) variance of supercoil angles of accordion-like fibrils in vessels that is furthe...
Evaluation of liver fibrosis: “Something old, something new…”
Annals of Gastroenterology, 2016
Hepatic fibrogenesis may gradually result to cirrhosis due to the accumulation of extracellular matrix components as a response to liver injury. Thus, therapeutic decisions in chronic liver disease, regardless of the cause, should first and foremost be guided by an accurate quantification of hepatic fibrosis. Detection and assessment of the extent of hepatic fibrosis represent a challenge in modern Hepatology. Although traditional histological staging systems remain the "best standard", they are not able to quantify liver fibrosis as a dynamic process and may not accurately substage cirrhosis. This review aims to compare the currently used non-invasive methods of measuring liver fibrosis and provide an update in current tissue-based digital techniques developed for this purpose that may prove of value in daily clinical practice.