David Sevrain

Mueller matrix polarimetry for improved liver fibrosis diagnosis

An experimental Mueller matrix polarimeter is used to quantify human liver fibrosis by measuring retardance and depolarization of thin biopsies. The former parameter is sensitive to fibrillar collagen, the latter is specifically sensitive to fibrillar collagen around blood vessels, which is not significant for liver fibrosis diagnosis. By using depolarization like a filter, retardance distribution enables distinguishing between disease stages and limits the high degree of observer discrepancy.

Two-photon microscopy of dermal innervation in a human re-innervated model of skin

When skin is injured, innervation can be severely disrupted. The subsequent re‐innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two‐photon excitation fluorescence (TPEF) microscopy of entire human skin explants re‐innervated by rodent sensory neurons labelled with the styryl dye FM1‐43. TPEF imaging of nerve fibres to a depth up to roughly 300 μm within the dermis was demonstrated, allowing three‐dimensional reconstruction of the neural tree structure. Endogenous second‐harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.

Evaluation of area-based collagen scoring by nonlinear microscopy in chronic hepatitis C-induced liver fibrosis.

In this paper we analyze a fibrosis scoring method based on measurement of the fibrillar collagen area from second harmonic generation (SHG) microscopy images of unstained histological slices from human liver biopsies. The study is conducted on a cohort of one hundred chronic hepatitis C patients with intermediate to strong Metavir and Ishak stages of liver fibrosis. We highlight a key parameter of our scoring method to discriminate between high and low fibrosis stages. Moreover, according to the intensity histograms of the SHG images and simple mathematical arguments, we show that our area-based method is equivalent to an intensity-based method, despite saturation of the images. Finally we propose an improvement of our scoring method using very simple image processing tools.

Measuring the scattering coefficient of turbid media from two-photon microscopy

In this paper, we propose a new and simple method based on two-photon excitation fluorescence (TPEF) microscopy to measure the scattering coefficient µ(s) of thick turbid media. We show, from Monte Carlo simulations, that µ(s) can be derived from the axial profile of the ratio of the TPEF signals epi-collected by the confocal and the non-descanned ports of a scanning microscope, independently of the anisotropy factor g and of the absorption coefficient µ(a) of the medium. The method is validated experimentally on tissue-mimicking optical phantoms, and is shown to have potential for imaging the scattering coefficient of heterogeneous media.

Snapshot Mueller polarimetry for biomedical diagnostic related to human liver fibrosis: evaluation of the method for biomedical assessments

Human liver biopsy samples, consisting into a 16 μm thickness biomaterial chemically fixed into a formaldehyde matrix, and stained by red picrosirius dye, are analysed for different states of fibrosis degeneration. Polarimetric methods, and specially Mueller polarimetry based on wavelength coding, have been qualified as an efficient tool to describe many different biological aspects. The polarimetric characteristics of the media, extracted from a Lu and Chipman decomposition1, 2 of their Mueller Matrix (MM), are correlated with the degeneracy level of tissue. Different works and results linked to the clinical analysis will be presented and compared to previous performed works.3 Polarimetric imaging will be presented and compared with SHG measurements. A statistical analysis of the distribution of polarimetric parameters (such as the retardance R and depolarisation Pd) will be presented too, in order to characterise the liver fibrosis level into the biomaterial under study.

PMO-150 Second harmonic generation microscopy of collagen and evaluation of liver fibrosis in chronic hepatitis C (CHC) infection

Introduction There is an urgent need to create tools to quantify collagen in liver fibrosis to facilitate stratification of disease and development of anti-fibrotic agents. Multiphoton microscopy enables imaging of unstained biopsies using endogenous sources of non-linear signals such as Two-Photon Excitation Fluorescence (TPEF) and Second Harmonic Generation (SHG). SHG allows specific detection of non-centrosymmetric structures such as fibrillar collagen, mainly type I. The SHG score is a measure of relative collagen area and is obtained by post-acquisition SHG/TPEF image processing. We have assessed the ability of our method to quantify collagen in advanced fibrosis due to CHC, with respect to Ishak stage (IS). Methods Biopsies from patients with advanced fibrosis (IS ≥3) were selected from 1 centre in the Trent Study of Patients with Hepatitis C Virus, a prospective cohort study formed in 1991. Index biopsies prior to 2008 were selected and notes reviewed for subsequent liver related outcomes (LRO). LRO was defined as variceal bleed, ascites, encephalopathy, HCC or liver related death. SHG was measured on 4μm FFPE sections. A mask of the biopsy area was created with TPEF. Image processing was performed by two independent researchers, blinded to Ishak stage, using in-house macros and each using different software (Image J & Matlab). PASW 17.0 was used for statistical analysis. Results The SHG score was acquired in 58 of 83 biopsies (66%). 25 were excluded due to signal artefact from paraffin, obscuring SHG signal from collagen. There was no significant difference in scoring by two researchers (p<0.001). The median SHG score was 15.96% (IQR 11.3–21.3%). Abstract PMO-150 figure 1 shows the median SHG score for each IS. SHG signal increased with disease severity (IS3:10.1%; IS4:14.1%; IS5:14.1%; IS6:21.2%). LRO occurred in 15 patients after a median of 57 months post-biopsy. The mean SHG score at index biopsy was 19.1% in those with, and 16.6% in those without subsequent LRO (non-significant difference, p>0.05).Abstract PMO-150 Figure 1 Conclusion SHG has proved to be a valuable method of quantifying collagen in liver fibrosis and does not require standard histochemical stains. Further development of this quantitative measure may result in a tool to assess response to anti-fibrotic therapy and progression to clinical endpoints. Competing interests None declared. References 1. Gailhouste L, et al. Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis. J Hep 2010;52:398–406. 2. Guilbert T, et al. A robust collagen scoring method for human liver fibrosis by second harmonic microscopy. Opt Expr 2010;18:25794–807.