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 &#176;C to 100 &#176;C; &#177;.01 &#176;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, &#177;2 &#176;C or &#177;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&#34; 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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...