In vivo label-free quantification of liver microcirculation using dual-modality microscopy

Abstract

Microcirculation lesion is a common symptom of chronic liver diseases in the form of vasculature deformation and circulation alteration. In acute to chronic liver diseases such as biliary atresia, microcirculation lesion can have an early onset. Detection of microcirculation lesion is meaningful for studying the progression of liver disease. We have combined wide-field fluorescence microscopy and a laser speckle contrast technique to characterize hepatic microcirculation in vivo without labeling in a bile-duct ligation rat fibrosis model of biliary atresia. Through quantitative image analysis of four microcirculation parameters, we observed significant microcirculation lesion in the early to middle stages of fibrosis. This bimodal imaging method is useful to assess hepatic microcirculation lesion for the study of liver diseases.

Fig. 1

Schematic illustration of optical set-up. For the wide-field fluorescence microscope, the light generated by mercury lamp passes through a single-band filter set (ET470/24x, 510dcxr, HQ525lp, Chroma Technology). The laser speckle contrast imaging uses a diode laser ($\lambda =638\mathrm{nm}$, 25-mW output) as the light source with an incident angle of about 30 deg. The resulting images were collected by a CCD camera.

Fig. 2

Wide-field fluorescence images and laser speckle contrast flow maps at different fibrosis stages. (a) Autofluorescence images; (b) laser speckle contrast flow maps, the blue color indicates faster speed, the red color indicates slower speed; (c) tissue sections stained with Hematoxylin/Eosin, black triangles indicate the proliferation of the biliary epithelia cells; (d) tissue sections stained with Masson Trichrome. Scale bar: $100\mu \mathrm{m}$.

Fig. 3

Quantification of four features extracted from the obtained autofluorescence images and laser speckle contrast images. With the progression of the liver fibrosis, (a) the number of junctions steadily decreases; (b) the average vessel breadth changes significantly only from stage 2 to stage 3; (c) the average vessel area decreases steadily; (d) the perfusion flow rate map increases drastically. All the results presented are normalized to the mean of the stage 0 (normal). ***$p<0.001$, **$p<0.01$, *$p<0.05$.

Fig. 4

The receiver operating characteristics curve analysis of the three selected features to demonstrate the performance of each feature in differentiating fibrosis stages.