title: Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.
abstract: OBJECTIVE
  Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD.
  
  
  METHODS
  We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals.
  
  
  RESULTS
  We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III.
  
  
  CONCLUSIONS
  This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.
authors.affiliation: Departments of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Juho
authors.initials: J
authors.lastname: Pirhonen
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Department of Pathology, HUSLAB, Helsinki, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Arola
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Sanja
authors.initials: S
authors.lastname: Sädevirta
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Panu
authors.initials: P
authors.lastname: Luukkonen
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, Biocenter Oulu, Oulu, Finland.
authors.forename: Sanna-Maria
authors.initials: SM
authors.lastname: Karppinen
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, Biocenter Oulu, Oulu, Finland.
authors.forename: Taina
authors.initials: T
authors.lastname: Pihlajaniemi
authors.affiliation: Departments of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Isomäki
authors.affiliation: Departments of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
authors.forename: Mika
authors.initials: M
authors.lastname: Hukkanen
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Hannele
authors.initials: H
authors.lastname: Yki-Järvinen
authors.affiliation: Departments of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland.|Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Elina
authors.initials: E
authors.lastname: Ikonen
mesh_terms: D000328:Adult; D005260:Female; D006801:Humans; D008103:Liver Cirrhosis / Q000473:pathology*; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D065626:Non-alcoholic Fatty Liver Disease / Q000473:pathology*