Brown adipose tissue (BAT) plays a key role in energy expenditure and heat generation Ardisiacrispin A and is a promising target for diagnosing and treating obesity diabetes and related metabolism disorders. fluorescence imaging of living mice shows extensive accumulation of the fluorescent probe in the interscapular BAT and ex vivo analysis shows 3.5-fold selectivity for interscapular BAT over interscapular WAT. Additional imaging studies indicate that SRFluor680 uptake is independent of mouse species and BAT metabolic state. The results are consistent with an unusual pharmacokinetic process that involves irreversible translocation of the lipophilic SRFluor680 from the micelle nanocarrier into the adipocytes within the BAT. Multimodal PET/CT and planar fluorescence/X-ray imaging of the same living animal shows co-localization of BAT mass signal reported by the fluorescent probe and BAT metabolism signal reported by the PET agent 18 The results indicate a path towards a new dual probe molecular imaging paradigm that allows separate and independent non-invasive visualization of BAT mass and BAT metabolism in a living subject. Keywords: brown adipose tissue molecular imaging in vivo fluorescence imaging PET imaging dual modality imaging Introduction Mammals utilize two types of adipose tissue for separate functions. White adipose tissue (WAT) stores excess triacylglycerols performs endocrine signaling and represents 20-25% of human body mass.1 Brown adipose tissue (BAT) confers adaptive thermogenesis and is 5% of the infant body mass. It is also found in adults but in much smaller quantities.2 3 BAT contains large amounts of mitochondria to dissipate chemical energy. Upon low temperature or pharmaceutical activation of the nervous system BAT uses high levels of uncoupling protein-1 to generate heat instead of ATP production. The tissue is highly vascularized which promotes efficient heat transfer to the bloodstream and body Ardisiacrispin A temperature maintenance.4 Medical imaging studies have shown that BAT mass is inversely correlated with body mass index and other obesity parameters in human adults.3 5 In recent years multiple animal model studies and early stage clinical trials have explored strategies to activate and increase BAT mass.6-10 Stimulation of BAT metabolism has been achieved through exercise low temperature and pharmaceutical agents. WAT can be transformed to BAT in a process known as “browning” using the same strategies.11-16 BAT mass has also been increased through transplantation with promising results.17 Notably BAT transplants have been reported to reverse type 1 diabetes by activating metabolism in the surrounding WAT.18 Most recently a clonogenic population of BAT stem cells has been identified in adult humans and SEL10 shown to functionally differentiate into metabolically active brown adipocytes.19 Brown and beige adipocyte-specific cell surface markers have been discovered which may serve as tools for the selective delivery of drugs.16 20 Taken together these emerging results suggest that BAT is a very promising imaging and therapeutic target for clinical treatment of obesity diabetes and related metabolic disorders.21 Biomedical research on BAT and clinical translation is facilitated by non-invasive imaging methods that visualize BAT in living subjects.22 The most common in vivo imaging technique uses 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) for positron emission tomography/computed tomography (PET/CT) of tissues with enhanced metabolism such as activated BAT.23 Several other nuclear probes have been investigated for BAT imaging and magnetic resonance imaging has also been utilized to monitor BAT morphology and chemical composition.24-30 While useful for the detection of human BAT Ardisiacrispin A which requires deep tissue imaging these Ardisiacrispin A methods employ radioactivity or expensive instruments and they are not convenient for preclinical research using small animal models. In contrast optical imaging of small animals is definitely highly attractive because it is definitely safe to perform relatively inexpensive and amenable to high Ardisiacrispin A throughput.31 The shallow interscapular location of BAT in mice is perfectly suited for optical imaging protocols.32 To day only two fluorescent molecular probes for BAT imaging have been reported; (1) IR786 a lipophilic cationic near-infrared dye with affinity for mitochondria 33 and (2) a fluorescently labeled nonapeptide that focuses on BAT vasculature.34 Both fluorescent probes are notable as pioneering lead molecular structures but they show modest in vivo imaging performance. The objective of this study was to find an.