This research was funded in part by a University of Connecticut Research Advisory Council Faculty Large Grant to M. Rubega. K. Burgio was supported on National Science Foundation NRT- IGE grant #1545458 to M. Rubega. The manuscript was significantly improved by the thoughtful feedback of two anonymous reviewers.

This work did not involve any work with live animals and was therefore exempt from animal care review.
1. Set-up and materials (Figure 1)
<ol>
	<li>If flat skins of the species of interest are not available, use Spaw&#8217;s protocol29 to create skins from fresh or frozen specimens. Preen feathers into a neat, natural position, and dry to obtain a constant weight before proceeding with measurements.</li>
	<li>Set up a constant temperature hot water bath. 
	NOTE: This set-up is quite tall, so it is easiest to place the hot water bath on the floor.</li>
	<li>Place a sheet of clear acrylic glass (Table of Materials) over the surface of the constant temperature hot water bath. The glass allows transmission of the heat to the underside of the skin without wetting the specimen. 
	NOTE: This pilot study used a sheet of acrylic glass (0.125 in thick). The thickness of the glass will not affect the emissivity30 of the material (it will always be 0.86), but it will affect the absolute temperature at the surface of the glass (i.e., thicker glass will result in a lower temperature). Thus, all measurements should be taken using an acrylic glass sheet of the same thickness.</li>
	<li>Place a piece of foam core board (1 in thick) with a circular hole (0.5 in diameter) over the acrylic glass. 
	NOTE: The size of the bird should guide the size of the hole and thus the size of uninterrupted feathering receiving heat from the source. Here, a 0.5 in diameter hole is used, because this size is large enough to achieve sufficient heat transmission to the specimen while still being small enough to center the heat under the breast feather tracts (of all birds except for the smallest ones). Regardless of the size of the thermal opening, to obtain a comparable and replicable value for every bird, make sure to perform measurements with holes of the same size.</li>
	<li>Attach a thermal camera to a tripod directly above the set-up at the camera&#8217;s minimum focusing distance. 
	NOTE: Here, a FLIR SC655 thermal camera is used (680 px x 480 px resolution, &#177;2 &#176;C or &#177;2% accuracy of reading, 40 cm minimum focusing distance). Other cameras may differ in degree resolution.</li>
	<li>Calibrate the camera by entering the following into the thermal camera software:
	<ol>
		<li>Find the reflected temperature by placing aluminum foil (shiny side facing up) over the foam with the emissivity value set to 1.0 in the camera calibration software. Take a thermal image. The temperature at the surface of the aluminum foil is the reflected temperature, which should be similar to the ambient temperature of the room.</li>
		<li>Set the emissivity value31 to 0.95. 
		NOTE: 1) Emissivity is the relative amount of heat that an object emits32 and ranges from 0 to 1. An object with a high emissivity value emits a large amount of heat, while an object with a low emissivity value radiates little heat32. This value represents the emissivity of feathers. 2) This value (0.95) is disputed. Cossins and Bowler claimed that feathers have an emissivity value between 0.90&#8211;0.95 but included no evidence31. Hammel reported a value of 0.98, but he obtained this value from a frozen specimen, so it may not be accurate33. Despite the lack of evidence, 0.95 emissivity is the value most often used in the thermal camera literature (as evidenced by Cossins and Bowler31).</li>
		<li>Make sure the ambient temperature and humidity in the room are constant. These values should be measured before every measurement and updated in the camera calibration software. The temperature and humidity of all indoor rooms will fluctuate somewhat, so recording these values and updating them in the software reduces measurement error. 
		NOTE: Here, FLIR ResearchIR Max software is used. This software does not save data for all images, so it is crucial to record all of these values for each image.</li>
	</ol>
	</li>
</ol>
2. Performance of measurements
<ol>
	<li>Set the constant temperature hot water bath to a target temperature (40 &#176;C is a proxy for the mean internal core temperature of most passerine birds)34. 
	NOTE: If working with a species whose resting core temperature is higher (e.g., hummingbirds34) or lower (e.g., penguins34 or ratites34,35), it may be appropriate to adjust the hot water bath accordingly. Figure 3 shows the relationship between the temperature of the hot water bath and temperature at the surface of the acrylic glass (e.g., the actual temperature of the heat source the flat skin is exposed to).
	<ol>
		<li>Results obtained from this protocol (see Figure 5) suggest that obtaining measurements across a range of temperatures is also informative of thermal performance differences. To accomplish this, follow the protocol using 5 &#176;C increments from 30&#8211;55 &#176;C.</li>
	</ol>
	</li>
	<li>In the thermal camera software, draw a circle/ellipse over the hole in the foam where the heat is escaping. This makes it possible to visualize this area when placing the skin on the foam to ensure that the correct area on the flat skin is measured.</li>
	<li>Place the flat skin specimen on the foam with the area of interest over the hole. 
	NOTE: Here, the belly region of each bird is measured, because it is not obstructed by other parts of the body such as the wing and is centrally located enough not to be subjected to edge effects. The placement of the skin over the heat hole will vary according to the experimental question. In general, placement directly over a feather tract, and as far away from the skin edge as possible, is recommended. Make sure not to flatten or disorder the feathers when placing the skin. If necessary, preen them into a natural position once the skin is placed.</li>
	<li>Wait 15 min to allow the skin to acclimatize to the heat source. If measurements are made too early, the temperature value at the surface of the feather coat will be too low. Here, the temperature transmission through the skin and feathers stabilized at 15 min, so waiting longer than 15 min will not yield an artificially high result.</li>
	<li>Taking a thermal image of the flat skin.
	<ol>
		<li>Set the emissivity value31 to 0.95 before taking the thermal image.</li>
	</ol>
	</li>
	<li>Remove the skin from the foam and immediately take a thermal image of the set-up without the flat skin on the foam. This quantifies the temperature at the surface of the acrylic glass and calibrates the area of the heat source with the measurement area on the flat skin.
	<ol>
		<li>Here, the emissivity of the acrylic glass used30 is 0.86. Make sure to record this in the thermal camera software before taking the picture without the skin. 
		NOTE: The temperature displayed by the hot water bath is not necessarily the temperature at the surface of the acrylic glass (Figure 3), since its thermal conductance is not perfect. Using the temperature of the glass reduces errors in estimating how warm the underside of the skin is, and is therefore a combined estimation of how much heat is lost through the skin and feathers.</li>
	</ol>
	</li>
	<li>Place the skin back on the foam in the same position. Repeat steps 2.5&#8211;2.6 for a total of five trials.
	<ol>
		<li>To place the specimen skin correctly, gently touch the feathers in the target measuring area with one fingertip, then remove the finger and view the thermal image. The residual heat from the finger will remain visible on the thermal image briefly. Check to ensure that the sampling area is within the visible circle drawn in the software, which represents the area of heat radiating through the skin from the hot water bath. If it is not, move the skin until it is. This process is illustrated in Figure 2. 
		NOTE: While fresh skins (when available) may represent the natural thermal performance of the skin in a living bird more closely, using dry skins for these measurements allow comparable, repeatable results on a much larger pool of specimens. Therefore, all measurements should either be taken using skins dried to constant weight, or on&#160;both the fresh and dried skin of specimens.</li>
	</ol>
	</li>
</ol>
3. Data collection from thermal images
<ol>
	<li>Each measurement consists of two thermal images: one of the flat skin and one of the acrylic glass. First, open the image of the acrylic glass. Align the circle drawn in the software with the hole in the foam visible in the image. Record the temperature value at the center of the circle. 
	NOTE: For more details on extracting data from thermal images, see Senior et al.36.
	<ol>
		<li>Make sure to calibrate the camera with the proper values. Set the emissivity30 to 0.86 and set the ambient temperature and humidity to match the current conditions in the lab, before recording the temperature value.</li>
	</ol>
	</li>
	<li>Open the thermal image of the flat skin. Without moving the circle, record the temperature value at the center of the circle. 
	NOTE: Because the circle is not recorded in the image, it is important to calibrate the placement of the circle with the image of the acrylic glass taken in section 2.6.
	<ol>
		<li>Make sure to calibrate the camera with the proper values. Set the emissivity31 to 0.95, and make sure to set the ambient temperature and humidity to the current conditions in the lab, before recording the temperature value.</li>
	</ol>
	</li>
	<li>Repeat steps 3.1&#8211;3.2 for all measurements of all specimens.</li>
</ol>
4. Calculation of thermal performance
<ol>
	<li>Subtract the temperature of the surface of the feather coat from the temperature of the acrylic glass. This value represents the heat retained by the feather coat.</li>
</ol>

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The authors have nothing to disclose.

This paper provides a protocol for repeatable, standardized thermal imaging measurements of avian flat skin specimens. This method makes it possible to compare thermal performance of the feather coat among species, within species, between comparable individuals, and at different locations on the bodies of individuals, all without destruction of the specimen.
The availability of necessary materials and equipment may be a limitation of this method. Although thermal cameras are rapidly becoming more accessible and affordable, research-grade thermal cameras still cost tens of thousands of dollars37. However, thermal cameras can be used for many practical applications in biology. McCaffery advocates for the use of thermal cameras to investigate ecological questions28. Thermal cameras are especially useful for collecting data on free-living organisms in the field because they are long-distance and non-invasive tools. The method presented here makes possible the integration of field and laboratory studies with measurements in the same units, as performed by the same equipment.
The use of thermal camera software other than that used here may necessitate modifications to this protocol, but such changes will only affect the set-up stage (section 1). Studies of smaller birds or certain questions about larger species of birds may require differently sized holes in the foam layer.
Similarly, the temperature of the hot water bath (step 2.1) may need modification for some species with higher or lower core temperatures if the goal is to measure temperatures with direct biological relevance to the experimental question. In general, water bath temperature standardization at 40 &#176;C across studies will facilitate comparative analysis of the relative thermal performance of different kinds of intact plumages and feather structures. If precise measurement of the energy flux across skin and feathers is required, the guarded hot plate method11,12,13,28 is likely a better approach, since it 1) eliminates air between the heat source and skin and 2) measures temperature at the inner surface of the skin directly. However, while this method does not measure or calculate energy transfer directly, it is designed to facilitate rapid and repeatable measurements of whole specimens. Finally, the results demonstrate ample precision in detecting patterns of variation in plumage thermal performance.
This method uses flat skins, which are not currently widely available in most museum collections. Round skins, which are abundantly available in most natural history collections, could be used with this method if demounted, softened, flattened, and redried. However, curators are unlikely to approve such remounting in most cases. To increase the resources for comparative studies of the thermal values of bird feathers, we advocate for widespread adoption of flat skinning in as many species as possible. Added benefits of flat skinning are that flat skins do not require the partial destruction of the skeleton and musculature of a specimen that round skinning does, and higher numbers of flat skins can be stored in the same space that round skins require.
In skins of any particular species, it is essential to develop a technique for accurately placing the skin in the same spot over the heat hole every time. The results obtained here suggest that the technique (as described in step 2.7) minimizes measurement error more rapidly and effectively than practice in skin placement alone. However, it is plausible that especially dense plumages (e.g., penguins11) may not lose sufficient heat through the feathers to make it possible to visualize heat holes through the skin and feathers on the thermal image.
Because of the presence of pterylae in most bird species, the arrangement of the feathers over the skin on a specimen will affect the pattern of heat transfer across the feather coat. Therefore, it is important that the feathers are positioned in as close to their natural position in a live bird as possible. Preening the feathers into a neat, natural position is the last step in the protocol for flat skinning a specimen29. Thus, if specimens are prepared properly, feather placement should be appropriate to species across specimens. The amount of ptiloerection of the feathers will also affect thermal performance of the feather coat by trapping insulating air in the feather coat. In contrast, in flat skinned specimens, the feathers lay flat on the skin29, so ptiloerection should effectively be comparable across all specimens.
Although this study focuses on birds, this method may be equally useful for mammal skins. Boonstra et al. asserted that bird feathers are more insulative than mammal fur, but this study was a qualitative assessment based on visual analysis of thermal videos39 rather than a quantitative measure of the heat escaping from comparable body areas. It is believed that the method described here will contribute to an expansion of comparative thermal research and yield great insight into the evolution and ecology of thermoregulatory structures such as feathers40.

Feathers are the defining characteristic of birds and serve many functions, among the most crucial being insulation1. Birds have the highest average core temperatures of any vertebrate group, and feathers insulating them from environmental temperature changes are a vital part of energy balance, especially in cold environments2. Despite the importance of feathers, the majority of literature on changes in thermal condition in birds has focused on metabolic responses to temperature variation rather than the function of feathers as insulation3,4,5,6,7,8,9,10 (for further details, refer to Ward et al.)11,12,13. However, feathers themselves may vary across time, individuals, and species.
The method presented here is useful for quantifying the overall thermal value of the feather coat alone. It removes confounding factors in live birds, such as behavioral thermoregulation1 and varying amounts of insulating fat. More widespread measurement of the thermal performance of feather coats is necessary to improve the understanding of how feathers contribute to insulation and how this varies among and within species throughout a bird&#8217;s life history and annual cycle.
Feathers insulate birds by trapping air both between the skin and feathers as well as within the feathers, and they create a physical barrier to heat loss14. Feathers consist of a central feather shaft, called a rachis, with projections called barbs14. Barbules are small, secondary projections on barbs that interlock with adjacent barbs together to &#8220;zip&#8221; up the feather and give it structure. Furthermore, down feathers lack a central rachis and have few barbules, therefore forming a loose, insulative mass of barbs over the skin14. Feather coats vary across species15,16, within species17,18, and within comparable individuals2,19,20,21,22,23,24. However, there is little quantitative information about how variations in number of feathers, the relative abundance of different types of feathers on a bird, or changes in the numbers of barbs/barbules affect the overall thermal performance of a feather coat. Previous studies have focused on determining a single mean value of insulation and thermal conductivity for a given species11,12,13.
The feather coat is known to vary among species. For example, most birds have distinct areas of skin from which feathers do, or do not, grow called the pterylae and apteria, respectively14. The placement of the pterylae (sometimes called &#8220;feather tracts&#8221;) varies across species and has some value as a taxonomic character14. However, some birds (i.e., ratites and penguins) have lost this pterylosis and have a uniform distribution of feathers across the body14. Additionally, different species, especially those inhabiting different environments, have different proportions of feather types15. For example, birds inhabiting colder climates have more down feathers15 and contour feathers with a larger plumulaceous portion16 than species inhabiting warmer environments.
The microstructure of certain types of feathers may also have an effect on insulation across species25,26. Lei et al. compared the microstructure of contour feathers of many Chinese Passerine sparrows and found that species inhabiting colder environments have a higher proportion of plumulaceous barbs in each contour feather, longer barbules, higher node density, and larger nodes than species inhabiting warmer environments25. D&#8217;alba et al. compared the microstructure of down feathers of common eiders (Somateria mollissima) and graylag geese (Anser anser) and described how these differences affect both the cohesive ability of the feathers and ability of the feathers to trap air26. Quantitative comparative data about how these variations in feathering affect the overall thermal performance of the feather coat across species is limited (for more details, refer to Taylor and Ward et al.)11,13.
Within a species, the feather coat&#8217;s thermal performance may vary. Some species, such as the monk parakeet (Myiopsitta monachus)17, inhabit very large and diverse geographical ranges. The different thermal stresses posed by these different environments may affect the feather coats of birds within a species regionally, but there are currently no data available on this topic. Additionally, Broggi et al. compared two populations of great tits (Parus major L.) in the northern hemisphere. They demonstrated that contour feathers of the more northern population were denser but shorter and less proportionally plumulaceous than those of the more southern population. However, these differences disappeared when birds from both populations were raised in the same place18.
Furthermore, Broggi et al. explained these findings as a plastic response to differing thermal conditions, but they did not measure the insulation values of these different feather coats18. The results also suggest that contour feather density is more important to insulation than the proportion of plumulaceous barbs in contour feathers, but Broggi et al. suggested that northern populations may be unable to produce optimal feathers due to a lack of adequate nutrients18. Quantitative measurements of the overall thermal performance of these feather coats would further the understanding of the significance of plumage differences.
Over time, the feather coats of individual birds vary. At least once a year, all birds molt (replace all of their feathers)19. As the year goes on, feathers become worn2,20 and less numerous18,21,22,23. Some birds molt more than once a year, giving them multiple distinct feather coats each year19. Middleton showed that American goldfinches (Spinus tristis), which molt twice a year, have a higher number of feathers and higher proportion of downy feathers in their basic plumage in winter months than they do in their alternate plumage during summer months24. These annual differences in the feather coat may allow birds to conserve more heat during colder periods passively or shed more heat passively during warm seasons, but no studies have tested this conclusively.
Although birds thermoregulate behaviorally1,27 and can acclimate metabolically to different thermal conditions3,4,5,6,7,8,9,10,26, feathers play an important role in thermoregulation by providing a constant layer of insulation. The method described here is designed to answer questions about the feather coat alone and its role in passive thermoregulation (i.e., how much heat does a living bird retain without modifying its behavior or metabolism?) by isolating the feathers. While active and physiological thermoregulation are ecologically important, it is also important to understand how the feathers alone aid in insulation and how they influence the need for active behavioral and physiological thermoregulation.
Previous studies have established methods for quantifying thermal conductivity and thermal insulation of animal pelts11,12,13,28. The method presented here is an extension of the &#8220;guarded hot plate&#8221; method11,12,13,28. However, the method described here measures the temperature at the outer boundary of the feather coat using a thermal camera, rather than thermocouples. The guarded hot plate method gives very precise estimates of energy flow through a pelt, but it requires construction of a multi-material hot plate, some familiarity with the use of thermocouples and thermopiles, and destructive use of a pelt that must be cut into small pieces. These pieces are then greased to eliminate air between the sample and hot plate apparatus. With the exception of the few birds that lack apteria (e.g., penguins), cutting small squares from bird skins is problematic for comparative purposes, since the location of the cut has large effects on the number of feathers actually attached to (and overlying) the skin. This problem is exacerbated by the variation among taxa in the presence, size, and placement of ptyerlae14.
Furthermore, while museum specimens can be a potentially rich resource for assessing the variation in insulation among birds, in general, permission to cut and grease skin specimens in scientific collections is unattainable. Additionally, specimens taken from the wild for guarded hot plate measurements cannot be subsequently used as museum specimens. The method presented here differs from the guarded hot plate method in that it can be used with whole dried bird skins, without 1) requiring the destruction of the specimen and 2) greasing the underside of the skin. It uses thermal cameras, which are increasingly affordable (though still relatively expensive), precise, and used for live bird measurements of thermal relations.
This method does not measure energy flow (and therefore thermal conductivity or insulation value) through the skin and feathers directly as the guarded hot plate method does. Instead, it measures the temperature at the outer boundary of a feather coat using a thermal camera. The resulting values represent an integrated measure of the heat lost passively through the skin, feathers, and air trapped between them (compared to a heat source underneath). Specimens prepared as flat skins and measured using the described technique can be stored in collections, and indefinitely provide value for future research. This method provides a standardized, comparable, and relatively simple way to measure feather coat thermal performance in any flat skinned specimen, which is especially useful in inter- and intra-specific comparisons.

<table><tbody><tr><td>Aluminum Foil</td><td>Reynolds Wrap</td><td>109000831</td><td>30 square ft.; this exact model need not be used.</td></tr><tr><td>Foam Core Board</td><td>Foamular</td><td>20WE</td><td>1 in. x 4 ft. x 8 ft; this exact model need no be used.</td></tr><tr><td>General Purpose Water Bath</td><td>PolyScience</td><td>WB02</td><td>Ambiet +5 &amp;deg;C to 100 &amp;deg;C; &amp;plusmn;.01 &amp;deg;C</td></tr><tr><td>PDF Data logger</td><td>Elitech</td><td>RC-51H</td><td>Built in temperature and humidity sensor</td></tr><tr><td>Plexiglass</td><td>AdirOffice</td><td>1212-3-C</td><td>Acrylic glass; 12 in. x 12 in. x 1/8 in.; this exact model need not be used.</td></tr><tr><td>Thermal Image Analysis Software</td><td>FLIR</td><td>ResearchIR Max v4.40.7.26 (64-bit)</td><td>Allows collection of precise, quantitative thermal data</td></tr><tr><td>Thermal Imaging Camera</td><td>FLIR</td><td>SC655</td><td>680x480-pixel resolution, &amp;plusmn;2 &amp;deg;C or &amp;plusmn;2% accuracy, 40 cm minimum focusing distance</td></tr><tr><td>Tripod</td><td>The Audubon Shop</td><td>The Birder Tripod with Manfrotto 700RC2 Rapid Release Head</td><td>65&quot; maximum height; this exact model need not be used.</td></tr></tbody></table>

using a thermal camera to measure heat loss through bird feather coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

Representative results from a series of one individual of each of five species, measured at six temperatures, are presented in Figure 4 and Figure 5. These show that small variations in the placement of the skin can result in variations in the readings of up to 1.7 &#176;C. Figure 4 shows how training of an investigator increases repeatability of the measurements. For example, the same individual house sparrow (Passer domesticus) was measured five times at a single target temperature by an inexperienced investigator (Figure 4A). After training on a variety of specimens of different species, one investigator (J.G.) measured the same specimen five times at the same target temperature (Figure 4B). The estimate of the relationship between the temperature of the acrylic glass and temperature at the surface of the feathers changed by only a small (but perhaps important) amount. As a result, repeatability of the measurements themselves changed by almost four-fold. Repeated practice is therefore highly recommended for operators on a non-sample skin (before taking measurements that will be analyzed) until measurements converge and variation in the measurements stabilize (i.e., no further improvement in reproducibility is seen with additional practice). This is important to achieve before conducting analyses on repeated measures (or the mean of repeated measures) in each specimen.
The data shown in Figure 5 represents a small pilot sample but suggests that this method for measuring thermal performance of the feather coat is likely to yield important insight into the thermal ecology of birds. To reduce measurement error, only one investigator (J.G.) trained and took the measurements. Although these data represent only a single individual of each of the species listed (house sparrow, eastern phoebe [Sayornis phoebe], gray catbird [Dumetella carolinensis], eastern bluebird [Sialia sialis], and tufted titmouse [Baeolophus bicolor]), variation in the slopes of the resulting data demonstrates that the thermal performance of feather coats varies among these individuals. Furthermore, the magnitude of these differences suggests that the variation may be due to species differences.
Moreover, given that a single trained investigator performed all the measurements in Figure 5, investigator skill alone does not control for variation in R2 values. For example, it was particularly difficult to obtain measurements repeatedly in the house sparrow, even after training, compared to the eastern phoebe and eastern bluebird (Figure 4, Figure 5). The latter two birds were both hatch-year individuals. Thus, their age class might influence the evenness of their insulation (although, that is speculation without further study), but there is no reason to expect that placement of their skins for the measurement should be any easier to repeat than that of the house sparrow. Thus, some incompletely understood quality of feather coats in the house sparrow may demand further investigation. Similarly, variation in slopes of the lines in Figure 5 suggests that measuring thermal performance across temperature ranges (e.g., the thermoneutral zone of a species) may be more biologically informative than using a single reference heat level.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60981/60981fig01.jpg" /> 
Figure 1: Diagram of the complete thermal camera and hot water bath set-up. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60981/60981fig01large.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/60981/60981fig02.jpg" /> 
Figure 2: Thermal images representing a method for replicating the same placement over multiple trials.&#160;The ellipse has already been placed on the heated area on the acrylic glass. These images show movement of the skin and not the ellipse. Gently and briefly touch a fingertip to the target measurement area on the feather coat. The fingertip will leave a heat mark on the skin for a few seconds. (A) Shown is the heat mark outside the ellipse, meaning that the target measurement area is not exposed to the heat. Being careful not to move the foam or acrylic glass (this would cause the ellipse to inaccurately represent the area of heat exposure), adjust the placement of the flat skin and touch the target measurement area again. Continue this process until (B) the heat mark is contained within the ellipse. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60981/60981fig02large.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/60981/60981fig03.jpg" /> 
Figure 3: Relationship between temperature of the hot water bath (display reading) and temperature at the acrylic glass surface (e.g., the actual heat source the flat skin is exposed to).&#160;It should be noted that the temperature at the surface of the acrylic glass is consistently slightly higher than the temperature displayed by the hot water bath. Use this figure only to gain an understanding of this relationship, and always measure the temperature at the surface of the acrylic glass for each trial (section 2.6). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60981/60981fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60981/60981fig04.jpg" /> 
Figure 4: Improvement in the repeatability of temperature measurements at the surface of a feather coat in a single bird.&#160;These values were obtained from an individual house sparrow (A) before and (B) after repeatability training by the investigator for measurement performance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60981/60981fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60981/60981fig05.jpg" /> 
Figure 5: Relationship between temperature at the acrylic glass surface and temperature at the feather surface in single specimens of five bird species.&#160;Points on any single graph represent repeated measurements in the same individual at six different target temperatures. It should be noted that although measurements at the reference heating point of 40 &#176;C are similar, the slope of these lines varies. This suggests that the thermal performance of feather coats in these birds differs (with a slope of 0 being a perfect insulator and slope of 1 being completely non-insulative). It should also be noted that measurement repeatability varies. Even after measurement training of the investigator, variance in repeated measures is highest for the house sparrow and lowest for eastern phoebe and eastern bluebird. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60981/60981fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats at JoVE.com

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

using-a-thermal-camera-to-measure-heat-loss-through-bird-feather-coats

Universidad San Sebastian

Research

JoVE Journal

Biology

3.4K Views.  University of Connecticut. This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Video: Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

Engineering

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 ...

Medicine

Characterization of Thermal Transport in One-dimensional Solid Materials

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including conductive, semi-conductive or nonconductive one-dimensional structures. This technique broadens the measurement scope of materials (conductive and nonconductive) and improves the accuracy and stability. If the sample (especially biomaterials, such as human head hair, spider silk, and silkworm silk) is not conductive, it will be coated with a gold layer to make it electronically conductive. The effect of parasitic conduction and radiative losses on the thermal diffusivity can be subtracted during data processing. Then the real thermal conductivity can be calculated with the given value of volume-based specific heat (&#961;cp), which can be obtained from calibration, noncontact photo-thermal technique or measuring the density and specific heat separately. In this work, human head hair samples are used to show how to set up the experiment, process the experimental data, and subtract the effect of parasitic conduction and radiative losses.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including ...

Measurements of Local Instantaneous Convective Heat Transfer in a Pipe - Single and Two-phase Flow

This manuscript provides step by step description of the manufacturing process of a test section designed to measure the local instantaneous heat transfer coefficient as a function of the liquid flow rate in a transparent pipe. With certain amendments, the approach is extended to gas-liquid flows, with a particular emphasis on the effect of a single elongated (Taylor) air bubble on heat transfer enhancement. A non-invasive thermography technique is applied to measure the instantaneous temperature of a thin metal foil heated electrically. The foil is glued to cover a narrow slot cut in the pipe. The thermal inertia of the foil is small enough to detect the variation in the instantaneous foil temperature. The test section can be moved along the pipe and is long enough to cover a considerable part of the growing thermal boundary layer.
At the beginning of each experimental run, a steady state with a constant water flow rate and heat flux to the foil is attained and serves as the reference. The Taylor bubble is then injected into the pipe. The heat transfer coefficient variations due to the passage of a Taylor bubble propagating in a vertical pipe is measured as function of the distance of the measuring point from the bottom of the moving Taylor bubble. Thus, the results represent the local heat transfer coefficients. Multiple independent runs preformed under identical conditions allow accumulating sufficient data to calculate reliable ensemble-averaged results on the transient convective heat transfer. In order to perform this in a frame of reference moving with the bubble, the location of the bubble along the pipe has to be known at all times. Detailed description of measurements of the length and of the translational velocity of the Taylor bubbles by optical probes is presented.

This manuscript describes methods aimed at measuring the local instantaneous convective heat transfer coefficients in a single or two-phase pipe flow. A simple optical method to determine the length and the propagation velocity of an elongated (Taylor) air bubble moving at a constant velocity is also presented.

This manuscript provides step by step description of the manufacturing process of a test section designed to measure the local instantaneous heat transfer ...

In Situ Surface Temperature Measurement in a Conveyor Belt Furnace via Inline Infrared Thermography

Measuring the surface temperature of objects that are processed in conveyor belt furnaces is an important tool in process control and quality assurance. Currently, the surface temperature of objects processed in conveyor belt furnaces is typically measured via thermocouples. However, infrared (IR) thermography presents multiple advantages compared to thermocouple measurements, as it is a contactless, real-time, and spatially resolved method. Here, as a representative proof-of-concept example, an inline thermography system is successfully installed into an IR lamp powered solar firing furnace, which is used for the contact firing process of industrial Si solar cells. This protocol describes how to install an IR camera into a conveyor belt furnace, conduct a customer correction of a factory calibrated IR camera, and perform the evaluation of spatial surface temperature distribution on a target object.

This protocol describes how to install an infrared camera into a conveyor belt furnace, conduct a customer correction of a factory calibrated IR camera, and evaluate the spatial surface temperature distribution of an object of interest. The example objects are industrial silicon solar cells.

Measuring the surface temperature of objects that are processed in conveyor belt furnaces is an important tool in process control and quality assurance. ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Neuroscience

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.

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 ...

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 ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Field-Based Thermal Physiology Assay: Cold Shock Recovery under Ambient Conditions

Ecological physiology, particularly of ectotherms, is increasingly important in this changing world as it uses measures of species and environmental traits to explore the interactions between organisms and their surroundings to better understand their survival and fitness. Traditional thermal assays are costly in terms of time, money, and equipment and are therefore often limited to small sample sizes and few species. Presented here is a novel protocol that generates detailed data on individual behavior and physiology of large, volant, terrestrial insects, using the example of butterflies. This paper describes the methods of a cold shock recovery assay that can be performed in the field under ambient environmental conditions and does not require costly laboratory equipment. This method has been used to understand the response and recovery strategy to cold shock of tropical butterflies, generating individual level data across entire butterfly communities. These methods can be employed in both remote field settings and classrooms and can be used to generate ecologically relevant physiological data and as a teaching tool.

Here, a low-cost, accessible protocol is described to evaluate cold shock recovery of butterflies under ambient environmental conditions.

Ecological physiology, particularly of ectotherms, is increasingly important in this changing world as it uses measures of species and environmental ...