the recent re - discovery of human brown adipose tissue ( bat ) ( 14 ) has triggered intense scientific interest in the potential of this tissue as a target against obesity and its metabolic abnormalities . numerous studies investigated the origin , plasticity , and metabolic characteristics of this tissue in animals ( reviewed at ( 5 ) ) . however , these findings must be translated into humans in order for any therapeutic value to be realized . currently , only a handful of studies have reported data from human bat ( 1 , 2 , 4 , 615 ) primarily using samples collected during surgeries ( 2 , 4 , 6 ) , necropsies ( 7 , 12 , 14 ) or using an open biopsy technique ( 1 , 8 , 9 , 13 ) . hampering the effort to study the biology of human bat is the lack of a safe method for the routine sampling of this tissue for research purposes . the bergstrm needle has long been used for non - image guided biopsy of muscles / soft tissues ( 16 ) , while computed tomography ( ct)-guided biopsy technique is well known and refined over the past 30 years for all types of tissues ( 17 ) . the purpose of this report is to describe a novel positron emission tomography ( pet ) and ct - guided bergstrm needle technique to sample supraclavicular human bat deposit . supraclavicular bat biopsies were obtained from healthy participants enrolled in clinical studies at the university of texas medical branch at galveston . informed written consent was obtained prior to inclusion in the studies from all participants in accordance with the declaration of helsinki . the institutional review board and the institute for translational science scientific review committee at the university of texas medical branch approved the experimental protocol . after 2 - 6h of non - shivering cold exposure ( 10 ) , a bolus injection of 185 mbq of 2-deoxy-2-[18f]fluoroglucose ( f - fdg ) was administered to participants . cold was used as a stimulus to activate bat thermogenesis and f - fdg uptake in bat . one hour later , a pet / ct scan ( general electric medical systems , milwaukee , wi ) was performed to visualize supraclavicular bat using the f - fdg - pet / ct images ( figure 1a ) . participants were transferred to the ct procedure room ( figure 1b ) and scout images of the lower neck ( figure 1c ) were taken to visualize the anatomy of the supraclavicular area ( general electric medical systems , milwaukee , wi ) . using major anatomical landmarks , an experienced interventional radiologist ( p.a . ) , identified the location of the supraclavicular bat depot in the ct images corresponding to the bat in the pet / ct scan . then , the skin over the area of the biopsy was thoroughly cleaned using chlorhexidine and covered with a sterile drape . a small amount ( 25 ml ) of 2% lidocaine was injected in the dermis of the biopsy site and in the subcutaneous adipose tissue depot . the injection needle was advanced in the targeted adipose tissue area and an additional ct scan was performed to ensure the correct placement of the needle at the biopsy cite , avoiding any adjacent superficial veins ( figure 1c ) . subsequent measures were taken to determine the depth of the bat deposit and the advancement of the biopsy needle . afterwards , an incision was made in the skin ( < 1 cm ) using a scalpel and a 6 mm bergstrm needle ( stille , lombard , il ) was inserted through the incision and advanced into the supraclavicular adipose tissue depot . once the needle was positioned appropriately , a trained member of the research team applied suction using a 60 ml syringe and a suction catheter ( figure 1d ) . precooled bacitracin ointment was applied to the top of the inner cannula to maintain suction and avoid leakage of air between the needle and the inner cannula . once the biopsy procedure was over , we applied manual compression for 35 min to minimize bleeding . subsequently , the incision was closed using standard 2.0 silk sutures and a sterile dressing was applied . the participants were instructed to avoid swimming and rigorous exercise for the first 72h after the biopsy . further , they were instructed to inspect the wound daily , apply antibiotic ointment once or twice a day and take painkillers for discomfort as needed . the participants returned to the clinical research center 58 days after the biopsy for suture removal . the clinical research notes and personal communication with the participants were used to evaluate the safety of the procedure and verify the incidence of adverse effects . complications were categorized to major and minor according to the clinical practice guidelines of the society of interventional radiology ( 17 ) . to establish our ability to sample the supraclavicular bat depot using this method , we conducted the following analysis in a supraclavicular bat and an abdominal subcutaneous white adipose tissue ( wat ) sample ( collected using the bergstrm technique needle ~10 cm right or left from the umbilicus ) using the following techniques : mitochondrial respiration : adipose tissue samples were immediately submerged in a cold ph adjusted preservation buffer and transferred to the laboratory analysis . respirometry was performed in an o2k oxygraph ( oroboros instruments , innsbruck , austria ) . briefly , bat and wat were weighed and suspended in the o2k chamber in 2ml of ph adjusted respiration buffer . respiratory capacity and function specifically , the ucp1 inhibitor gdp ( 30 mm final concentration ) was titrated into the o2k chamber to confirm the presence and quantify the function of ucp1 . u6382 , sigma - aldrich , st louis , md , abc kit and dab kit vector laboratories , inc , burlingame ) were used in protocol . an olympus microscope ( bx41c ) with cellsens viewer 1.5 software ( olympus soft imaging solutions gmbh , munster , germany ) was used for slide imaging . gene expression : rna was extracted from adipose tissue using a pure link rna isolation mini kit total ( life technologies , carlsbad , ca ) . cdna was synthesized by high - capacity rna - to - cdna kit ( life technologies , carlsbad , ca ) and pre - amplified by taqman preamp master mix kit ( life technologies , carlsbad , ca ) . quantitative real - time - pcr analyses were performed on an abi prism 7900ht using the taqman gene expression master mix ( life technologies , carlsbad , ca ) , pre - amplified cdna and specific taqman gene ucp1 expression assays ( hs00222453_m1 , life technologies , carlsbad , ca ) . gapdh ( hs02758991_g1 ) was used as the housekeeping gene to normalize the expression of the target gene . the study sample consisted of 23 men , 443 years old , bmi 292 kg / m who underwent a total of 43 supraclavicular biopsies . on one occasion ( 2.3% of biopsies ) , the participant developed a subcutaneous hematoma , which fully resolved . on four occasions ( 9.6% of biopsies ) , the participants complained of numbness and tingling in the site of the biopsy , which resolved without further complaints . the amount of supraclavicular bat obtained using this pet and ct - guided needle biopsy was 100200 mg of tissue . the amount was enough to conduct quantitative pcr , immunohistochemistry , and mitochondrial respiration analyses . analyses conducted in one sample confirm that tissue collected had characteristics similar to animal bat : ( 1 ) the gene expression of uncoupling protein 1 ( ucp1 ) was 61 times higher than that of abdominal subcutaneous wat from the same subject ; ( 2 ) histology results show minimal ucp1 staining ( 0.99% strong positive ) in the subcutaneous abdominal wat ( figure 2a ) and intense ucp1 staining ( 22% strong positive ) in the supraclavicular bat ( figure 2b ) ; ( 3 ) the supraclavicular bat samples had a higher uncoupled respiration compared to the abdominal adipose tissue in basal conditions and upon the addition of substrates ( figure 2c  d ) ; and ( 4 ) in the supraclavicular bat sample , o2 consumption decreased following the addition of the ucp1 inhibitor gdp , indicating the presence of ucp1 ( figure 2c ) ( 20 ) . the proposed method is of significance because it can be safely used to sample the supraclavicular bat deposit for research purposes . the majority of previous studies that have sample the human bat used either postmortem specimens ( 7 , 12 , 14 ) or samples obtained during surgeries ( 2 , 4 , 6 ) . however , the fact the subjects sampled were either deceased or had an underlying pathological condition , may limit the generalizability of the results . other studies conducted a limited number of open biopsies ( 1 , 8 , 9 , 13 ) . although open biopsy techniques can also be used to biopsy human bat , they are more invasive than the proposed technique . to the best of our knowledge , this is the first report to describe and evaluate the safety of a minimally invasive method to routinely sample the supraclavicular human bat depot . further , this method yields adequate tissue for molecular and functional analysis of the tissue without damaging its morphological characteristics . limitations of this method include its high cost , exposure to radiation , and the need for equipment and adequately trained personnel . adaption of this method from investigators in the area of bat research may increase the availability of human bat specimens and accelerate the development of current scientific knowledge .