brown adipose tissue ( bat ) has received renewed interest for its potential to reduce adiposity and improve metabolic health . while bat was traditionally considered to be lost after infancy , recent evidence supports the presence of functional bat in adult humans [ 24 ] . bat is unique in its expression of uncoupling protein-1 ( ucp1 ) , which is activated by cold and -adrenergic signaling as a means to maintain core body temperature in cold environments . when activated , ucp1 facilitates proton leak across the mitochondrial inner membrane to dissociate oxygen uptake from atp synthesis and generates large amounts of heat . to this end , numerous regulatory , transcriptional and endocrine factors are being investigated and developed to augment bat mass and/or activity in order to increase energy expenditure through enhanced ucp1 activation . therefore non - invasive , objective assessments of bat activity have become increasingly necessary to determine whether adipose tissue thermogenic capacity has been altered by various interventions in pre - clinical rodent models . several nuclear imaging methods have been employed to measure bat activity in rodents , such as pet / ct [ 710 ] , fmri , or c - mrs   that provide estimates of adipose tissue metabolism . however , these methods rely on the assumption that circulating substrate uptake ( fdg - pet / ct , c - mrs ) or blood flow ( fmri ) reflects bat activity . since thermal energy is a specific end product of ucp1-mediated metabolism in bat , its detection is an ideal surrogate of bat activity . several studies have measured surface body temperature using infrared imaging in rodents as an analog to bat activity under basal or stimulated conditions [ 1418 ] , however , it remains unknown whether this technique truly measures bat - derived thermogenesis . therefore , we chose to test whether surface body temperature assessed using infrared thermography accurately reflects changes in bat activity in  vivo when challenged with the highly specific 3-adrenoreceptor agonist cl-316,243 , which is greater than 10,000 times more selective over other -adrenergic receptors . in order to test the specificity of our protocol ( figure  1a and b ) to detect ucp1-derived thermogenesis , we injected mice lacking ucp1 ( ucp1 ) and wild - type litter mates ( ucp1 ) with saline or the 3-adrenergic agonist cl-316,243 on separate days and measured oxygen uptake and dorsal surface temperature . ucp1 mice had a significant increase in oxygen uptake following injection of cl-316,243 compared with the saline trial ( 50    8% higher , figure  1c , p  <  0.05 ) , however , this effect was absent in ucp1 litter mates ( 7    3% higher , p  >  0.05 ) . similarly , only ucp1 mice increased dorsal surface temperature in the interscapular ( figure  1d , p  <  0.05 ) or entire body area ( figure  1e , p  <  0.05 ) , demonstrating that the thermal changes detected using the infrared camera were specific to ucp1 ( for representative images see figure  1f ) . we next sought to determine whether infrared thermography could be used to detect differences in bat activity in response to ambient housing temperature . the thermoregulatory adaptation of bat activity in colder environments is considered one of the main functions of bat . we found that mice housed at 23  c had a greater rate of oxygen consumption with cl-316,243 and basally compared with mice bred and housed at 30  c ( figure  2a , main effect of housing temperature and cl-316,243 treatment as well as an interaction effect , p  <  0.05 ) . the surface temperature of the interscapular roi showed similar changes to vo2 with the 3-agonist ( figure  2b , main effect of housing temperature and cl-316,243 treatment as well as an interaction effect , p  <  0.05 ) , but the body roi only showed a main effect of housing temperature and cl-316,243 treatment ( figure  2c , p  <  0.05 , for representative images see figure  2d ) . due to the agreement between vo2 and surface temperature in the above experiments , we determined the association between the cl-316,243 induced change in oxygen uptake and the corresponding change in surface body temperature using the interscapular and whole body rois . we also included male mice housed at 23  c in this analysis in order to determine if this relationship was independent of gender . we found significant associations between changes in oxygen uptake and dorsal surface temperature of the interscapular region ( figure  2e , r  =  0.602 , p  <  0.05 ) and the whole body ( figure  2f , r  =  0.507 , p  <  0.05 ) , demonstrating a strong positive relationship between changes in whole body energy metabolism and thermogenesis detected via infrared imaging . the present results demonstrate for the first time that stimulated dorsal surface temperature represents an accurate determination of ucp1-mediated thermogenesis . additionally , we show this measurement is sufficient to differentiate between mice maintained under standard animal housing conditions ( 23  c ) , a stimulus known to promote a thermogenic program in adipose tissue , compared with mice raised at thermoneutrality ( 30  c ) . a benefit of the present method is that it causes minimal physiologic stress in the tested animals . previous studies examining responses to stress with thermal imaging have shaved rodents prior to testing , which may alter the natural thermoregulatory state of the animals and preclude other subsequent experiments . other studies have used infrared imaging to measure bat activity in conscious mice   or using non - selective -adrenergic agonists   without validating that the resulting heat signature is derived from bat . moreover , under these conditions it is very likely that non - adipose tissues such as skeletal muscle may influence changes in whole body thermogenesis , confounding the interpretations of results . further , the described analysis of thermal images can be done quickly , is relatively low cost , and does not require highly trained personnel as compared with nuclear imaging . in addition , compared with fdg - pet there is no radiation , which allows sequential and more frequent measurements in the same animal . traditional measures of bat metabolism utilize the principle of local glucose uptake ( fdg - pet ) or blood flow ( fmri ) as analogous measures of tissue activity . however , fdg uptake in response to cl-316,243 stimulation remains inducible in female mice that lack ucp1 , demonstrating some degree of non - ucp1 related glucose metabolism . further , adipose tissue vascular function is reduced with insulin resistance , therefore , local blood flow may not accurately reflect adipocyte metabolism . it should be noted that a recent study utilizing mrs imaging of infused c - pyruvate may address some of the pitfalls of fdg - pet / ct and mri imaging , however , to our knowledge this method has not been performed in ucp1 mice to demonstrate its selectivity for bat activity . in the future it will be necessary to compare thermal imaging with other methods used to detect bat activity following different metabolic stimuli . the current method uses dorsal thermal imaging and since in mice bat is located in the interscapular region , it is likely that bat is the predominant contributor to the heat generated after an adrenergic challenge . further validation using thermal imaging of mice on the ventral plane is required to determine whether this technique may be sensitive enough to detect changes in the activity and/or abundance of beige / brite adipose tissue . brown adipose tissue holds an enormous potential to improve metabolic health . in order to more accurately determine the capacity for bat activation , we have validated the use of infrared thermography as a means to rapidly assess bat - derived thermogenesis . mice heterozygous for ucp1 expression were obtained from jackson laboratories and bred to produce wild - type ( ucp1 ) and ucp1-null ( ucp1 ) mice . mice were bred and housed at an ambient temperature of 30  c to prevent thermal stress to the ucp1 mice . a separate cohort of eight male and eight female c57bl6 mice were acquired from jackson laboratories at 11 weeks of age and were housed at 23  c . all cohorts of mice were tested at 34 months of age , maintained on a chow diet ( harlan teklab diet # 8640 ) , and kept on a 12  h light / dark cycle ( lights on at 0700h ) . mice were handled according to procedures previously approved by the mcmaster university animal research ethics board . for the metabolic cage studies , mice were tested on two days separated by at least 24  h  in a room maintained at 2628  c . injection of 0.5  mg / g body weight avertin ( 2,2,2-tribromoethanol dissolved in 2-methyl-2-butanol ; sigma aldrich ) . after 2  min , mice were i.p . injected ( opposite side of the body as anesthetic ) with saline or 0.033  nmol / g  body weight of the 3-adrenergic agonist cl-316,243 ( sigma aldrich ) dissolved in saline . this dose of cl-316,243 was chosen as it is low enough to not produce secondary effects such as inflammation in white adipose tissue . mice were then placed dorsal side up onto an enclosed stationary treadmill and oxygen uptake was monitored using an indirect calorimetry system ( columbus instruments , columbus , oh ) with air sampled every 5  s. twenty minutes following anesthetic injection , mice were removed from the metabolic cage and placed dorsal side up on a bare cage grid . this prolonged period following injection of saline or cl-316,243 allows for stabilization of oxygen uptake ( vo2 ) , maximal activation of bat ( in the case of cl-316,243 ) and consistent thermal measurements ( figure  s1a ) . an infrared camera ( t650sc , emissivity of 0.98 , flir systems ) mounted in the top of a box placed overtop of the mouse on the cage grid was used to acquire a static dorsal thermal image at a focal length of 30  cm immediately after removal from the metabolic monitoring treadmill . mice were tested with saline and cl-316,243 on separate days rather than sequentially on the same day because we found in pilot experiments that , while measurements of oxygen uptake resembled the current data , thermal imaging data failed to show changes with cl-316,243 ( figure  s1b  d ) , likely due to body cooling in the unconscious mice . thus , one can only compare thermal measurements if the anesthetic cooling effect is standardized across treatments . indirect calorimetry measurements were exported to csv files using the clams examination ( clax ) software . vo2 measurements for each condition are the average of the final 5  min  of measurement . in order to analyze thermal images , csv files using the flir research ir program , and then batch converted to . raw files . the .raw files were imported into the freely available image analysis software amide for mac osx ( http://amide.sourceforge.net/index.html ) by specifying the data format as float , little endian 32-bit , 0 offset bytes , 480    640    1 dimensions and gate , frames , and voxel size as 1 . images were aligned and overlapped in order to draw a region of interest ( roi ) capturing the entire body of the mouse ( body roi ) or a smaller area surrounding the interscapular region ( interscapular roi ) such that each type of roi was overlaid on all thermal images at once . this allowed for identical sizing of each type of roi ( body box or interscapular box ) across all mice and conditions . the interscapular roi was sized 2.07  cm by 2.28  cm for all mice , started at the rearward base of the ears ( figure  1b ) and was centered laterally on the scapulae . the body box was sized 9  cm by 4.2  cm in this study in order to accommodate the body of the largest mouse . the absolute scaling of the images was confirmed using the cage grid spacing as the standard . for each roi , the mean of the warmest 10% area was calculated using amide and used as the primary variable across conditions . using the mean of the highest 10% area allows for an objective quantification of thermal changes , as the warmest part of the torso of wild - type mice is always the interscapular region in wild - type mice . temperature scaling of representative thermal images was centered around the mean body temperature of all mice being compared and a scale span of 4  c was used for good color contrast between the saline and cl-316,243 treatments . for analyses comparing ucp1 and ucp1 mice or mice housed at 23  c and 30 c a 2-way repeated measures anova was used to assess genotype / housing temperature and stimulation condition effects . when significant differences were observed ( p  <  0.05 ) , a tukey 's honestly significant different post - hoc test was used to assess specific differences .
the activation and expansion of brown adipose tissue ( bat ) has emerged as a promising strategy to counter obesity and the metabolic syndrome by increasing energy expenditure . the subsequent testing and validation of novel agents that augment bat necessitates accurate pre - clinical measurements in rodents regarding the capacity for bat - derived thermogenesis . 
 we present a novel method to measure bat thermogenesis using infrared imaging following 3-adrenoreceptor stimulation in mice . 
 we show that the increased body surface temperature observed using this method is due solely to uncoupling protein-1 ( ucp1)-mediated thermogenesis and that this technique is able to discern differences in bat activity in mice acclimated to 23  c or thermoneutrality ( 30  c ) . 
 these findings represent the first standardized method utilizing infrared imaging to specifically detect ucp1 activity in  vivo .