title: Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.
abstract: All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
authors.forename: Suman
authors.initials: S
authors.lastname: Ranjit
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Dobrinskikh
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: John
authors.initials: J
authors.lastname: Montford
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dvornikov
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Lehman
authors.affiliation: Department of Pathology, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Orlicky
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: Raphael
authors.initials: R
authors.lastname: Nemenoff
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA. Electronic address: egratton@uci.edu.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address: moshe.levi@ucdenver.edu.
authors.forename: Moshe
authors.initials: M
authors.lastname: Levi
authors.affiliation: Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Furgeson
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005355:Fibrosis; D007668:Kidney / Q000473:pathology*; D051379:Mice; D008856:Microscopy, Fluorescence; D009400:Nephrosclerosis / Q000175:diagnosis*; D061848:Optical Imaging
keywords: FLIM; SHG; UUO; autofluorescence; collagen; fibrosis