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.   Significance:  Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network.   Aim:  Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution.   Approach:  By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways.   Results:  The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the “minimum work” hypothesis of the vascular network fitting to Murray’s law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation.   Conclusions:  Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.

Three-dimensional vascular and metabolic imaging using inverted autofluorescence