Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images.

Suman Ranjit,Moshe Levi,Enrico Gratton,Alexander Dvornikov,Seth Furgeson

doi:10.1364/boe.7.003519

Abstract

Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of fibrosis with time is detected using this multiparametric approach to phasor analysis.

Highlights

IntroductionPhasor approach to fluorescence lifetime imaging has been applied to study cellular metabolism and to evaluate presence of reactive oxygen species in living cell and tissue samples [1,2,3,4,5,6]
Phasor approach to fluorescence lifetime imaging has been applied to study cellular metabolism and to evaluate presence of reactive oxygen species in living cell and tissue samples [1,2,3,4,5,6]. This approach is well established for mapping lifetime to a fluorescence image and separating areas of image having distinctively different lifetimes. We have applied this phasor approach to study a combination of lifetime imaging and second harmonic imaging to decipher the extent of fibrosis of the kidney in an unilateral ureter obstruction (UUO) model [7,8,9,10]
3.1 FLIM and phasor signature of the UUO samples Fluorescence lifetime images having an image size of 360 μm were obtained for each animal

Summary

Introduction

Phasor approach to fluorescence lifetime imaging has been applied to study cellular metabolism and to evaluate presence of reactive oxygen species in living cell and tissue samples [1,2,3,4,5,6]. The FLIM data acquisition is independent of the direction of the acquisition (contrary to the detection of harmonics) and can be obtained from any laser scanning microscope equipped with FLIMBOX [14] or other fluorescence lifetime measuring cards and this method is applicable in more commonly available instruments This technique offers a new method where diseased and control tissues are separated and the effect of therapeutic interventions can be measured. The main difference of this approach compared to the SHG generation or immunohistochemistry is that this technique does not involve either tissue modification or a specialized instrument This is a new analysis method which uses FLIM images from any laser scanning microscope capable of fluorescence lifetime imaging and analyzes the phasor distribution originated from the FLIM image to distinguish control and diseased tissues. The paper uses UUO model as a proven model of fibrosis [14] and as a system to show the methodology of this multiparametric analysis when control and diseased sets are known

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