This study was funded by the Croatian Science Foundation research grant (IP-2018-01- 7416).

All experimental procedures on laboratory animals were approved by the National Ethical Committee and the Ministry of Agriculture (EP 185/2018). The experiments were conducted in accordance with the Ethical Codex of The Croatian Society for Laboratory Animal Science and ARRIVE guidelines. All procedures performed in studies involving human participants were in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Zagreb, School of Medicine (UP/I-322-01/18-01/56). In this study, we present the results from three female participants (BMI: 29 kg/m2 &#177; 5 kg/m2). Informed consent was obtained from all human volunteers for their participation in the study and for presenting the data.
1. Measuring the activation of brown adipose tissue after a meal in humans
NOTE: Perform the experiments during the summertime when the daily temperature is not below 22 &#176;C to keep basal BAT activity as low as possible.
<ol>
	<li>Carefully choose the control healthy participants (if BAT activity is to be estimated under pathological conditions) since BAT activity is gender, age, BMI, and even the phase of estrous cycle dependent.
	<ol>
		<li>To estimate the phase of the menstrual cycle of female participants, ask them questions about how long their average menstrual cycle is and the date of the first day of their last menstruation. Do not forget to mark the date of the experiments. 
		NOTE: Proper selection of matching control subjects is the hardest part of clinical studies since healthy controls and participants with pathological conditions should be as similar as possible and differ only in the disease investigated.</li>
	</ol>
	</li>
	<li>Ask the participants to rest well, not to have breakfast (fasting-no intake of calories), to gather in the morning hours for the experiments, and to rest for at least 30 min to avoid possible BAT activation during muscle activity via sympathetic activation.</li>
	<li>Ask the participants to remove their upper clothing 15 min before the measurements to avoid the possible effects of warming the skin surface (thermal effects of clothes) while determining the baseline BAT activity. Perform measurements at appropriate room temperature (22-27 &#176;C).</li>
	<li>Perform infrared measurements.
	<ol>
		<li>While the participants are resting, mount the thermal camera (detector type: uncooled microbolometer; detector pitch: 17 &#181;m; camera spectral range: 7.5-14.0 &#181;m; thermal sensitivity: 20 mK at 30 &#176;C; lenses: 36 mm; resolution: 1024 pixels x 768 pixels; instantaneous field of view [IFOV]: 0.47 mRad) on the tripod and position it 1 m away from the spot where the participant will be seated. 
		NOTE: If the measurements are performed in colder weather (outside air temperatures below 15 &#176;C at 50% humidity), put the camera at room temperature and turn it on for at least 1 h before performing the measurements. Cold instruments can give different results after warming up to room temperature due to autocalibration.</li>
		<li>Connect the thermal camera to a computer and software as instructed by the manufacturer. Record aluminum foil (crumpled then stretched aluminum foil) at a focal distance of 1 m to determine the reflected temperature of the room, presented as measured temperature. In the camera software, input the distance of 0 m and emissivity of 1. 
		NOTE: The reflected apparent temperature is a parameter obtained when the emissivity of the camera is set to 1.0 and the distance to 0 m and the measurements are taken on crumpled then stretched aluminum foil. Reflected apparent temperature represents an approximation of the total incident infrared radiation on the detector from the environment.</li>
		<li>Just before starting the measurements, determine the room air temperature and air humidity (necessary for later analysis). Instead of taking one thermal image, record a movie. From the movie, later choose the best possible image frame for analysis to reduce the possibility of losing valuable data.</li>
		<li>Before starting the recording, set the following parameters: the duration of video recording at 10-15 s (or any other desired value), the frame rate at 5 fps (frames per second) or any other value (in our hands, 5 fps is the maximum needed), and a location on the disk where the movie will be saved, as described below.
		<ol>
			<li>In the software above the main camera window, choose the third icon from the left. In the pop-up menu, choose Edit Record Settings, after which a new window will open.</li>
			<li>In record mode, select Record to Disk, and below that, set the Record for This Duration at the desired time. In the record options of the same window, limit record rate to 5 (Hz) and choose the location where the recordings will be saved.</li>
			<li>To set the frame rate, close the existing window, open Edit in the main menu, and select Preferences. In the right part of the opened window, enter 5 in Target Frame Rate. In the same window below, select Hotkey/Remote Start Can Stop Record, and from the drop-down menu, select In Start/Stop mode. 
			NOTE: Try to make the shortest possible movies with the lowest possible frame rate since it is memory-consuming. At these settings, one record will have approximately 100 Mb.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Position the participant so that the supraclavicular area of the neck, above the collarbone where BAT is located (Figure 1), is at a 1 m focal distance and record a short movie (10-15 s) at a frame rate of 5 fps by pressing the F5 key. The recording will stop at the designated time.</li>
	<li>In the room at the time of the measurements, ensure only the participant and the person who is performing the measurements are present. Avoid air movement or draft (e.g., from air conditioning). Ensure the participants are away from cold draft, sunlight (direct or indirect), or any source of heat like light bulbs.</li>
	<li>If suitable, measure the blood glucose concentrations in capillary blood from the fingertip with a standard glucometer and the body temperature using an axillary thermometer.</li>
	<li>Ensure all the participants eat the same meal. Pay attention to the food restrictions and requirements of the tested subjects (for example, the meal for diabetic patients). All participants, including control (healthy) people and participants with metabolic disorders, should eat the same meal. 
	NOTE: For more details about meals that diabetic patients can consume, contact a local endocrinologist or discuss it with the participants suffering from diabetes mellitus.</li>
	<li>At the desired time after a meal, make the new recording by pressing F5 using the same setting values. Do not repeat the set protocol for recordings. Repeat the measurements at 30 min, 1 h, 2 h, and 3 h after a meal12. For your specific study design, the time after a meal could be shorter or longer, but we recommend at least the first three time points. 
	&#8203;NOTE: The limitation of the number of participants is four to six, even though the measurements are done fast. With a higher number of participants, the delay time for some will be too long.</li>
</ol>
2. Measuring the activation of brown adipose tissue after a meal in laboratory animals
NOTE: Since the animals are housed in an animal facility with regulated room temperature and a day/night cycle of 12 h/12 h the experiments could be performed during any season. The room temperature during experiments should be between 22 &#176;C and 27 &#176;C. In this study, six female animals in diestrus and six male wild-type (WT) C57Bl/6NCrl animals were examined.
<ol>
	<li>Anesthetize the animals as per the institution&#39;s ethical guidelines. In this study, anesthesia was performed using i.p. injections of ketamine/xylazine (80-100 mg/kg and 6-8 mg/kg, respectively). Apply eye gel to both eyes to prevent corneal drying during anesthesia. Shave the interscapular regions of the test animals a day before the experiments (the region of the skin between the shoulder blades) using a small animal trimmer.</li>
	<li>The day before the experiments, also determine the phase of the estrous cycle in female animals. 
	NOTE: The phase of the estrous cycle is determined by vaginal smears.
	<ol>
		<li>Dip a cotton tip swab in room-temperature sterile saline solution (0.9% NaCl) and insert it into the vagina. Gently scrape the vaginal wall with the swab, spread the attached cells onto a glass slide, and allow it to air dry.</li>
		<li>Put the animals back in their cages. Stain the cells with 500 &#181;L of 0.1% cresyl violet acetate for 1 min, after which rinse them 3 times with water.</li>
		<li>View the cells under a light microscope with 100x magnification and bright-field illumination. Determine the phase of the estrous cycle based on the number of leukocytes and nucleated and cornified epithelial cells observed in the smear25.</li>
	</ol>
	</li>
	<li>Remove the animals&#39; food the evening before the experiments (fasting overnight) with water ad libitum. The best way is to transfer the animals to new clean cages to avoid possible remnants of food in the cages.</li>
	<li>On the morning of the experimental day, prepare the thermal camera and recording settings as done for testing the human participants.</li>
	<li>Do not disturb or cause stress to the animals before performing IR measurements. Carefully place the animal in a clean cage (ensures there are no effects of other animals&#39; scent on the animal&#39;s sympathetic system). Place the cage under the thermal camera at a focal distance of 1 m. Record a movie by pressing F5.</li>
	<li>Weigh the food pellet before giving it to each animal so that food intake can be calculated. Allow the animal to eat for 30 min in its cage and weigh the food pellet again after the meal. In this study, female animals ate 0.038&#160;&#177; 0.004 g food/body weight. 
	NOTE: If you decide to measure blood glucose concentrations, perform the measurements before a meal but after IR measurements to ensure that this will not lead to BAT activation by the sympathetic system.</li>
	<li>Repeat IR measurements at the desired time after starting a meal (usually 30 min, 1 h, and 2 h after a meal)17,26.</li>
	<li>After all the experiments are completed, test again the phase of the estrous cycle in female animals as described above (female animals may exit the desired phase of the estrous cycle sooner than anticipated).</li>
</ol>
3. Analyzing the thermal recordings
NOTE: The thermal camera software calculates the object&#39;s temperature using five variables.
<ol>
	<li>Set the following variables in the software before analysis: skin emissivity, e = 0.9815,27, reflected room temperature (as calculated from the image of aluminum foil), air temperature, relative humidity, distance to the object = 1 m. Perform the analysis by using the software with these values. 
	NOTE: The preferred color palette is rainbow since it uses more hues, which allows for easier detection of BAT above the collarbone.</li>
	<li>For each movie, enter the listed variables into the camera software on the right side of the main window. Select the suitable frame (image) from the movie by moving the play head at the bottom of the screen or pressing the Pause button.</li>
	<li>Select the region of interest (ROI) by choosing the desired shape of the area on the left side of the main window. Choose the shape that best corresponds to the area of skin above or between the collarbones.</li>
	<li>When the ROI is chosen, the minimal, maximal, and average temperatures of the ROI are displayed on the right side. In the image, the red triangle represents the point of maximal recorded temperature, and the blue triangle represents the minimal recorded temperature. Repeat this step for several frames to be sure that the measured temperature is stable during a few seconds of the recording.</li>
	<li>Subtract the maximal temperatures of the skin area above BAT before a meal from the maximal temperatures after a meal to determine the increase in postprandial BAT activity in laboratory animals.</li>
</ol>

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The authors have nothing to disclose.

Recent studies present growing evidence regarding the physiological regulation and importance of BAT activity in adult humans and animals in the development of obesity and diabetes mellitus. Furthermore, possible BAT activation by exogenous activators is becoming a target for pharmaceutical companies. To be able to estimate the physiological regulation and pathophysiological importance of BAT in very burdensome diseases, as well as discover a potential therapeutic approach, infrared thermography is becoming the method of choice. Even though the IR technology is emerging as a new method of choice in measuring BAT activity12,13,14,15,17, special attention should be paid not to the applied method itself but to the physiological attributes of BAT activation. One should pay special attention to gender, age, phase of the estrous cycle, feeding status, and possible stress in human participants and laboratory animals.
Measurements of the increase of BAT activity after a meal performed on humans and laboratory animals are quite similar12,17. Critical steps in conducting these measurements include the avoidance of stress and muscle activity (animals should not be disturbed before performing measurements). For female participants, the functional differences in BAT activity during the estrous (menstrual) cycle should be taken into consideration. Furthermore, the skin should be bare (animals should be shaved the day before experiments are performed to avoid unnecessary stress). Furthermore, it is very important to set the camera software properly to get the best thermal resolution possible. To present the data as thermal images, the best way is to choose a proper palette of coloring that suits the particular study. If the protocol is followed properly, there is less possibility of making errors in performing IR thermography.
The limitation of the method is that it is not possible, without a slight concern, to compare the BAT activity between people with different quantities of subcutaneous fat (or obese animals to animals with normal body weight). To avoid this problem, the same subject should be used as the control by measuring the BAT activity before and after a meal. The calculation of the increase in BAT activity after a meal will eliminate the differences in heat distribution from the subcutaneous BAT to the skin surface.
Reporting BAT activity through changes between the maximal skin temperatures of both skin areas is, in our point of view, a better way since it eliminates the differences in BAT size and/or designated skin area in the results gained by average temperature measurements29.
As mentioned earlier, the method of choice for measuring BAT activity to date is PET-CT using radioactive glucose (18F-FDG). This method has, over time, proven to be not sensitive enough, its usage of radioactive material is concerning, and it is very expensive. PET-CT is useless in determining BAT activation after a meal. 18F-FDG accumulation in BAT after a meal is barely visible10 and considered a negative result. Recently, it was suggested that BAT activation is more pronounced in the human population and, therefore, is increasingly important in the development and spread of metabolic diseases7. An attempt to solve this problem is to measure the BAT volume by MRI in prediabetic and patients with T2DM with insulin resistance11. However, the amount of BAT does not give any information about the activity of BAT and the utilization of glucose and fatty acids, which is important in diabetic patients.
Establishing IR thermography as a method of choice for measuring BAT activity, especially in human subjects with high blood glucose concentration, will have a huge impact on the field. IR thermography is used for the determination of cold-induced activation of BAT13,14,15 and, recently, BAT activation after a meal in healthy volunteers12 and laboratory animals17. The postprandial BAT activation is smaller in females and older subjects no matter the species. Unfortunately, the majority of research on BAT activation and metabolic studies altogether have been performed only on male subjects (male laboratory animals or men). The problem with this approach is that it ignores the effect of the phase of the estrous cycle on BAT activity30.
Finally, IR thermography will enable the possibility of investigating the physiological and pathophysiological importance of BAT in different human populations, especially in pre- and postmenopausal women, studies for which are missing.

Brown adipose tissue (BAT), in contrast to white adipose tissue (WAT), does not store but rather spends energy. Upon sympathetic stimulation, BAT utilizes fatty acids and glucose and produces heat by the activation of uncoupling protein 1 (UCP1). The function of UCP1 is to use an H+ gradient between two mitochondrial membranes to produce heat instead of ATP. The function of BAT is to increase heat production under cold conditions, which leads to an increase in energy expenditure1. After cold exposure, sensory inputs from the skin inhibit warm-sensitive neurons in the median preoptic (MnPO) nucleus of the hypothalamic preoptic area (POA), which diminishes the inhibitory effect of POA neurons on the rostral raphe pallidus (rRPa). The activation of rRPa neurons increases sympathetic activity, which is followed by an increase in BAT activity2,3. Cold-induced BAT activation improves insulin sensitivity in humans4, and this activity is decreased in humans with increased body mass index (BMI) and age1,5,6,7.
Apart from its role in cold-induced thermogenesis, after a meal, glucose uptake in the BAT increases in the lean male population, contributing to diet-induced thermogenesis (DIT), which is higher in BAT-positive male subjects8,9. The state-of-the-art technique used for measuring BAT activity is positron emission tomography computed tomography, known as PET-CT. This method determines BAT activity by measuring the accumulation of the radiotracer fluorodeoxyglucose (18F-FDG). However, PET-CT fails as the method of choice for detecting the activation of BAT after a meal. One of the reasons is that, after a meal, 18F-FDG competes with postprandial hyperglycemia for the same glucose transporter, which makes it unsuitable for determining BAT activation after a meal, especially when comparing BAT activity in healthy and diabetic participants with possible differences in blood glucose concentrations. Furthermore, BAT uses fatty acids as a source of energy for heat production which is not visible with PET-CT. 18F-FDG accumulation in BAT after a meal is barely visible10 and is, therefore, interpreted as a negative result in most cases. Unsurprisingly, recently, it was suggested that the activation of BAT is more pronounced in the human population than we had previously thought; therefore, a new approach to detect BAT activity and its involvement in metabolic disorders is necessary7. An attempt to solve this problem is to measure the volume of BAT with magnetic resonance imaging (MRI) in prediabetic patients and patients with diabetes mellitus type 2 (T2DM) with insulin resistance11. However, BAT volume measured by MRI is not a sufficient indicator for estimating the everyday function and usage of glucose and fatty acids by BAT. Therefore, to estimate real differences in BAT activity in healthy versus T2DM patients, a new approach is needed that offers a possibility to find out the pathological mechanism of BAT malfunction in T2DM patients.
To determine the activation of BAT, we performed measurements of BAT heat production before and after a meal using infrared (IR) thermography (Figure 1)12,13. Establishing IR thermography as a method of choice for measuring BAT activity after a meal in healthy and obese individuals or patients with diabetes mellitus will have a huge impact on the field. To this day, IR thermography is used for the determination of cold-induced activation of BAT13,14,15. In recent human history, cold-induced BAT activity is not very pronounced anymore (due to proper heating of habitats, proper clothing), while BAT activation after a meal occurs every day. Furthermore, the physiological regulation of these two BAT functions via the hypothalamus is completely different. After a meal, the activation of proopiomelanocortin (POMC)-expressing neurons in the hypothalamic arcuate nucleus (Arc) leads to an increase in sympathetic nerve activity via rRPa16. Cold-induced activation of BAT measured by IR thermography or PET-CT is improper when used as a measure for everyday BAT activity. Increased BAT activity after a meal is followed by glucose utilization, which is ultimately important for maintaining glucose homeostasis, insulin sensitivity, and the daily regulation of glucose concentration. Postprandial BAT activation leads to an increase in postprandial glucose consumption, followed by an increase in heat production and body temperature (DIT). This was shown to be gender, age, and BMI dependent12. Similar gender differences in BAT activation after a meal are observed in male and female laboratory mice17. These findings correspond to recently discovered gender differences in the regulation of BAT by Burke et al., who showed that the hypothalamic regulation of BAT browning via a subpopulation of POMC neurons differs in male and female mice18. The postprandial activation of BAT is smaller in women, older populations, and obese people. The lack of BAT activation after a meal (decreased glucose utilization) could lead to a higher prevalence of impaired glucose tolerance in women19,20,21,22. Unfortunately, the majority of studies on BAT activation were done only on men. By activating BAT after a meal, glucose uptake increases in the lean male population. It is not surprising that, after BAT activation, DIT is higher in BAT-positive male subjects8,9. Furthermore, BAT transplantation in male mice improves glucose tolerance, increases insulin sensitivity, and decreases body weight and fat mass23.
PET-CT fails as a method of choice for measuring BAT activity, especially after a meal. Therefore, a non-invasive and more sensitive method was developed. IR thermography enables the estimation of BAT activity in different laboratory animals (knock-out mice), as well as human participants, regardless of gender, age, or the effects of different pathological conditions on BAT activity. An additional benefit of this method is the simplicity for participants and laboratory animals, which allows us to estimate the potential benefits of BAT booster therapy. The recent studies using IR thermography for determining the physiological behavior of BAT after cold exposure or a meal are described in the recent publication of Brasil et al.24.

<table><tbody><tr><td>0.1% cresyl violet acetate&amp;nbsp;</td><td>Commonly used chemical</td><td></td><td></td></tr><tr><td>Device for measuring air temperature and humidity</td><td>Kesterl</td><td>Kestrel 4200</td><td>Certificat of conformity</td></tr><tr><td>External data storage</td><td>Hard Drive with at least 1 TB</td><td></td><td></td></tr><tr><td>Glass microscopic slides</td><td>Commonly used</td><td></td><td></td></tr><tr><td>Small cotton tip swab&amp;nbsp;</td><td>Urethral swabs</td><td></td><td></td></tr><tr><td>Software for analysis</td><td>FLIR Systems, Wilsonville, OR, USA</td><td>FLIR Tools</td><td></td></tr><tr><td>Software for meassurements</td><td>FLIR Systems, Wilsonville, OR, USA</td><td>ResearchIR software</td><td>FLIR ResearchIR Max, version 4.40.12.38 (64-bit)</td></tr><tr><td>Thermac Camera</td><td>FLIR Systems, Wilsonville, OR, USA</td><td>FLIR T-1020</td><td></td></tr></tbody></table>

infrared thermography for the detection of changes in brown adipose tissue activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

The easiest way to determine BAT activity is to subtract the maximal skin temperature above the BAT before and after a meal in human subjects. A better way to calculate BAT activity is to select two regions of interest: the skin area above the BAT, which is located in the supraclavicular area, and the interclavicular area of the skin where no BAT tissue is found in humans, designated as a reference area (according to PET-CT; Figure 1). BAT activity is then easily determined by the subtraction of those two temperatures. As shown in Figure 1, BAT activity was determined as 1.8 &#176;C. To determine the activation of BAT after feeding, this was repeated at designated time points after a meal12. In this study, we performed experiments with three female participants. Similar results were obtained from participant 1 and participant 2 with BMIs of 23 kg/m2 and 34 kg/m2, respectively. Participant 3 had the lowest BMI (18 kg/m2) and the highest increase in BAT activity after a meal (Figure 1B, left). The maximal temperature on the supraclavicular area of the skin is not a good representation of BAT activity since there is no decrease in skin temperature even 3 h after a meal (Figure 1B, right). Due to differences in BAT activity among the human population, the analysis of the results should be done individually, and/or choosing the participant for the study should be done very carefully. There is not such a difference in BAT activity in laboratory animals since they are closely related.
The first advantage of this method compared to the use of PET-CT (18F-FDG) is that it measures BAT activity irrespective of the source of heat. For that reason, IR thermography is much more sensitive and represents more truthfully the BAT activity under different physiological or pathophysiological conditions. This method can be used to determine the physiological conditions that lead to changes in BAT activity, such as age, gender, or phase of the estrous cycle. In humans, an added benefit is the ability to determine the pathophysiological changes in BAT activity in metabolic diseases like obesity and diabetes mellitus type 2. A special benefit could be seen when comparing the BAT activity of people with high blood glucose concentration to healthy controls since, in PET-CT, the radioactive glucose competes with the same glucose transporters as glucose in the body, leading to false negative results or misrepresentations of BAT activity.
One possible concern is the differences in the thickness of the skin, especially the subcutaneous white adipose tissue, which can alter the skin temperature above BAT in different subjects28. This problem can be avoided by comparing the BAT activation before and after a meal. Usually, the activity and following importance for different pathophysiological conditions are determined by BAT activation after cold exposure. However, this type of BAT activation is seasonal and, in humans (compared to animals in wilderness), not very important. To determine the effects of BAT activity on everyday life, as well as glucose homeostasis, BAT activation after a meal followed by glucose and fatty acid utilization is the proper way.
Attention should be paid to the measurements performed in warm surroundings or too soon after muscle activity. Muscle activity increases body temperature, which leads to vasodilatation in the skin (Figure 2). When there is no visible warmer skin area above the BAT compared to the surrounding skin areas, exclude the recordings from the study.
BAT activity in laboratory animals is determined by the maximal temperature of the BAT located between the scapula (interscapular BAT, iBAT) and the average temperature of the skin above the iBAT. It is easier to measure the average temperature of the skin above BAT in laboratory animals since it is more localized than in humans (Figure 3). In this study, the changes in BAT activity after a meal were measured in female WT animals in diestrus (28.2 &#177; 0.5 weeks old). As shown in Figure 3, statistically significant changes in BAT activity were determined 30 min after a meal but only when the maximal temperature was measured (p &#60; 0.05). When the BAT activity was presented as changes in average temperatures, the change was not significant (p = 0.066). Therefore, presenting the BAT activity via changes in the maximal temperature of the interscapular skin area is a better way to present the results. This change in BAT activity was positively correlated with the amount of ingested food (r = 0.65).
The major concerns for both set of subjects are the biological differences in BAT activation in male and female and young and old animals, as well as the differences in BAT activity during the estrous cycle17. Special attention should be paid to the amount of time passed from the last meal, which will be hard to estimate if the above protocol is not followed. Performing experiments at the same time in the morning is not precise enough17.
An additional concern while measuring BAT activity is to avoid stress in the animals as much as possible. Any disturbance will increase sympathetic activity and, therefore, BAT activity16. Furthermore, most mistakes involving temperature recordings may appear if the interscapular area of the mice is not shaved properly, which may lead to measuring the wrong area of the skin.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64463/64463fig01.jpg" /> 
Figure 1: Infrared thermography. (A) Two skin areas are selected: one above the brown adipose tissue (BAT; supraclavicular area) and the second one in the interclavicular area (no BAT tissue present below the skin of this area-reference point). The BAT location is presented as the warmest area above the collarbone. The schematic representation of the BAT location in the neck according to PET-CT is presented in the left figure. Temperatures are maximal temperatures measured in encircled skin areas. The bar represents 5 cm. (B) The differences between maximal temperatures in both skin areas are presented for each participant, showing the increase in BAT activity 2 h after a meal (left). BAT activity cannot be presented by maximal temperatures of the skin above BAT (right). Results are presented as mean &#177; standard error of the mean. * p &#60; 0.05 compared to starting value (paired ANOVA). Abbreviation: SC = suprascapular. <a href="https://www.jove.com/files/ftp_upload/64463/64463fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64463/64463fig02.jpg" /> 
Figure 2: The conditions when BAT activity cannot be measured. The figure represents the conditions when it is not possible to determine BAT activity due to increased vasodilatation of the skin blood vessels. The bar represents 5 cm. <a href="https://www.jove.com/files/ftp_upload/64463/64463fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64463/64463fig03.jpg" /> 
Figure 3: Brown adipose tissue in laboratory mice. (A,B) Interscapular brown adipose tissue (BAT) is activated after cold exposure and only then shown as 18F-FDG accumulation by PET-CT. The activity of BAT could be seen with a thermal camera without cold exposure. (C) The location of the BAT in the thermal scan corresponds to the BAT presented in the PET-CT image by an arrow. The shaved area is bigger than the BAT to be able to see all the changes in temperature (the location of Tmax could not be perfectly predicted, and in some studies, the average temperature can be measured). The bar represents 1 cm. (D) The measured maximal and average temperatures of the skin above BAT for six female mice in diestrus are presented. Results are presented as mean &#177; standard error of the mean. * p &#60; 0.05 compared to starting value (paired ANOVA). <a href="https://www.jove.com/files/ftp_upload/64463/64463fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity at JoVE.com

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

infrared-thermography-for-detection-changes-brown-adipose-tissue

Nanyang Technological University

Research

JoVE Journal

Medicine

1.9K Views.  MKP Ltd.. Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Video: Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Behavior

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biology

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

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