A novel approach to the quantification of hepatic stellate cells in intravital fluorescence microscopy of the liver using a computerized image analysis system
Xy. Zhang et al., A novel approach to the quantification of hepatic stellate cells in intravital fluorescence microscopy of the liver using a computerized image analysis system, MICROVASC R, 60(3), 2000, pp. 232-240
Hepatic stellate cells (HSC) are nonparenchymal liver cells which reside in
the space of Disse within the hepatic microcirculatory unit. HSC can be di
stinguished using intravital fluorescent microscopy (IVFM) due to the autof
luorescence from their intracellular vitamin A. Herein we report on a novel
approach for the quantification of video-recorded rat HSC images acquired
by IVFM (excitation 360 nm/ emission 420 nm) by the combined use of the "Ce
ll counting macro" and the "Measurement macros" in the NIH image software.
The approach involved two major steps using (i) the "Cell counting macro" f
or automatic detection, threshold-setting, and generation of a binary image
of the vitamin A autofluorescence in the HSC images and (ii) the "Compute
percent black and white" command in the "Measurement macros" to automatical
ly determine the HSC density (%), which was then expressed as percentage of
the total area of vitamin A autofluorescence-associated sites per observat
ion area. Comparing the vitamin A autofluorescence areas in the original an
d the binary fashion HSC images revealed that the "Cell counting macro" was
an optimal option for the analysis of the low-magnification (x10 objective
) HSC images, whereas this macro was not suitable for the analysis of the h
igher magnification (40x objective) HSC images unless modifications were ma
de. Our analysis revealed that HSC represent approximately 4-5% of the tota
l area of the liver surface. In analyzing the higher magnification HSC micr
ofluorographs, the use of the original "Cell counting macro" resulted in a
significant underestimation of HSC density (60% reduction, P < 0.01) when c
ompared with those analyzed using our modified macro. This study represents
the first report of an automatic and reliable approach to the intravital f
luorescent microscopic quantification of HSC using a computer-NIH image ana
lysis system. (C) 2000 Academic Press.