Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis

Suman Ranjit; Evgenia Dobrinskikh; John Montford; Alexander Dvornikov; Allison Lehman; David J Orlicky; Raphael Nemenoff; Enrico Gratton; Moshe Levi; Seth Furgeson

doi:10.1016/j.kint.2016.06.030

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis

Kidney Int. 2016 Nov;90(5):1123-1128. doi: 10.1016/j.kint.2016.06.030. Epub 2016 Aug 21.

Authors

Affiliations

¹ Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
² Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
³ Department of Pathology, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA.
⁴ Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA. Electronic address: egratton@uci.edu.
⁵ Department of Medicine, University of Colorado-Anschutz Medical Campus, Aurora, Colorado, USA. Electronic address: moshe.levi@ucdenver.edu.

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.

Keywords: FLIM; SHG; UUO; autofluorescence; collagen; fibrosis.

Publication types

Validation Study

MeSH terms

Animals
Disease Models, Animal
Fibrosis
Kidney / pathology*
Mice
Microscopy, Fluorescence*
Nephrosclerosis / diagnosis*
Optical Imaging*

Abstract

Publication types

MeSH terms

Grants and funding