The authors thank Perkin Elmer Company for supporting this publication. We also thank Alana Ross, B.S. for proofreading this manuscript.

Note: All animal studies should be performed under approved institutional protocols and animal care guidelines.
1. In Vivo CLI imaging BAT with 18F-FDG
1.1 Background Imaging:
<ol>
	<li>Before 18F-FDG injection, place four nude mice in an induction chamber that has been connected with isoflurane balanced with oxygen for 5 min to induce anesthesia. Then place the four anesthetized mice into the imaging apparatus.</li>
	<li>Acquire the background image with the following parameters: Open filter, f = 1, bin = 8, FOV = D, and exposure time = 120 sec, stage temperature = 37 &#176;C.</li>
</ol>
1.2 BAT Imaging with 18F-FGD:
<ol>
	<li>Inject the anesthetized mice with 280 &#181;Ci of 18F-FDG in PBS intravenously in the tail vein. After the injection, return the mice to a cage equipped with food and water. 
	Note: It is necessary to intravenously inject 18F-FDG to achieve a decent contrast around BAT area. Only a very weak contrast can be seen with the same amount of 18F-FDG if an intraperitoneal injection is used.</li>
	<li>Acquire CLI images at 30, 60, and 120 min after 18F-FDG injection with the same parameters as background imaging (step 1.1.2). For each imaging session, allow 5 min for the anesthesia reinduction.</li>
	<li>To quantify the signal to noise ratio (S/N), use the imaging software interface to draw two equal sized ellipse ROIs over the interscapular BAT and an area adjacent to the BAT (reference area) (Figure 1b).</li>
</ol>
1.3 Validation of the CLI Source:
<ol>
	<li>Anesthetize four mice and inject the mice with 280 &#181;Ci of 18F-FDG intravenously.</li>
	<li>Sacrifice the mice 60 min after the injection of 18F-FDG by injection of sodium pentobarbital (200 mg/kg, IP). Carefully remove the skin from the interscapular area. Image all of the mice with the same parameters as above (1.1.2), at the same time (Figure 2a).</li>
	<li>Carefully remove the interscapular white adipose tissue (WAT) and BAT, and then image the dissected mice with the same parameters (Figure 2b).</li>
	<li>From the CLI images, calculate the contribution from BAT by using two ROIs with the following equation: R(BAT) = (CLIa-CLIb)(BAT)/(CLIa-CLIb)(BAT-removed), where ROIa is for the interscapular area and ROIb is for the reference area (Figure 2b).</li>
</ol>
2. BAT Imaging Application
2.1 Monitoring Activation with NE
<ol>
	<li>Divide nude mice into two groups (n = 4 each).</li>
	<li>Inject one group with norepinephrine (NE) (50 &#181;l, 10mM) intraperitoneally. Use the second group as a non-activated control. After 30 min, anesthetize both groups with isoflurane for 5 min, and then intravenously inject each mouse with 18F-FDG (220 &#181;Ci). 
	Note: The intraperitoneal injection of NE should be 30 min before 18F-FDG injection to avoid dramatically agitating the mice.</li>
	<li>Image the mice 60 min after 18F-FDG injection using the same parameters as the above protocols.</li>
</ol>
2.2 Monitoring Activation Under Cold Exposure
<ol>
	<li>Measure the body temperatures of the mice in the cold room with a rectal thermometer. Temperatures should be about 30 &#176;C.</li>
	<li>Image the mice 60 min after 18F-FDG injection by using the same imaging parameters as the above protocols. Use mice that are kept at room temperature (25 &#176;C) as the controls, and image them with the same.</li>
	<li>For a cold exposure study, place the mice in a cold room (4 &#176;C) for 4 hr before 18F-FDG injection and return the mice to the cold room after each imaging session and after recovering from anesthesia.</li>
	<li>parameters as above.</li>
</ol>
2.3 Monitoring Deactivation of BAT Under Long Anesthesia
<ol>
	<li>For long anesthesia (60 - 70 min), inject mice intraperitoneally with ketamine/xylazine and keep them under anesthesia for 60 - 70 min at room temperature.</li>
	<li>Once the mice are anesthetized, inject 18F-FDG (220 &#181;Ci) intravenously via the tail vein.</li>
	<li>Image the mice 60 min after 18F-FDG injection by using the same parameters as the above protocols.</li>
</ol>
3. Spectral Unmixing and Multispectral Cerenkov Luminescence Tomography Studies
<ol>
	<li>Anesthetize a mouse for 5 min, and then inject 300 &#181;Ci 18F-FDG intravenously. Acquire multispectral images 60 min after 18F-FDG injection from the animal&#8217;s dorsal side with the following parameters: f = 1, bin = 16, acquisition time = 300 sec per filter, emission filters = 580, 600, 620, 640, 660 and 680 nm.</li>
	<li>Conduct spectral unmixing with Living Imaging 4.3.1 software and select two components (BAT and unspecific signals) and automatic unmixing (Figure 4).</li>
	<li>Conduct the 3D reconstruction according to the method reported by Kuo et al. 28,43. Incorporate the Cerenkov emission spectrum into the diffuse light propagation model, apply Tikhonov regularization in the NNLS (nonnegative least squares) optimization of the residuals, and generate the surface tomography (which is used for 3D image coregistration) from the structure light imaging (Figure 5). 
	Note: For multispectral CLI spectral unmixing and tomography, at least 5 images with different filters are needed.</li>
</ol>

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</ol>

Publication of this video-article is supported by&#160;Perkin Elmer Company.

Research related to BAT has been conducted for several decades. Previously, it had been considered to have no significant physiologic relevance during human adulthood 1. However, recent large scale clinical PET imaging with 18F-FDG and other investigations have confirmed BAT is still present in the upper chest, neck and other locations in adults 2,3. Recent studies have strongly suggested that BAT plays an important role in obesity and diabetes 2,6. Other research also indicates that BAT plays important roles during the process of ageing 12 and that its activity could be enhanced through exercise 10,44.
Both in clinical human studies and preclinical research, PET imaging with 18F-FDG is the most used method to study BAT. However, for preclinical animal studies, PET is generally much more expensive than optical imaging. In this protocol, we demonstrate that CLI with 18F-FDG can be applied for optically imaging BAT in small animals. Like other optical imaging techniques, tissue penetrating limitation and low sensitivity for deep targets are the intrinsic limitations of CLI 25,32. Nonetheless, recently Spinelli et al. demonstrated that decent CLI signal could be observed with 10 mm tissue depth with 32P 28,43, and Thorek et al. showed that 16 mm penetration could be achieved in lymph nodes of patients after 18F-FDG injection 39,40. Compared to PET imaging, CLI can be performed with a relatively low cost imaging system. Recently, Thorek et al. demonstrated that significantly high sensitivity could be achieved with CLI as low amounts of 18F-FDG accumulated (about 2Ci) in the nodes of patients 39,40. In vitro testing indicated that CLI signal from 0.01 Ci 90Y was detectable in solution 25,32,33. Moreover, CLI also has the potential for high spatial resolution and the capacity for high throughput screening. In addition, CLI is easy to learn and use.
In the course of experiments, we found that CLI contrast of BAT with 18F-FDG is highly related to the injection methods. Intravenous injection of 18F-FDG can provide excellent contrast for interscapular BAT, while intraperitoneal injection with the same amount of 18F-FDG only showed a weak contrast in the BAT area.
Spectral unmixing, a very practical technique to separate two sets of signals, has been widely used in fluorescence imaging. It is well known that the uptake of 18F-FDG is not highly target-specific, and different targets/tissues have different light attenuation properties. We found the peak of the CLI spectrum of BAT is around 640 nm, and this data reflected the actual contexts of BAT. The 3D reconstruction of this protocol is very operable, because it can be performed with a commercially available optical imaging system based on the light diffusive properties of different wavelengths. With this approach, we can avoid the use of a specialized imaging system that requires multiple-angle view images for 3D reconstruction.
For both spectral unmixing and 3D reconstruction, a greater minimum of 600 photon counts from each image in BAT is required. To this end, large binning (bin = 16), small f stop (f = 1) and a long acquisition time (5 min) are needed for each image.
In summary, harnessing the unique location and shape of BAT and the significantly high uptake of 18F-FDG in BAT, we demonstrate how BAT in small animals can be optically imaged with 18F-FDG via the CLI technique. This method can be reliably used for imaging BAT and for monitoring BAT activation. Additionally, we also demonstrate that spectral unmixing and 3D reconstruction are practicable and 3D volume quantification is possible for future studies.

Brown adipose Tissue (BAT) is a special tissue for thermogenesis in mammals, and one of its major functions is to maintain the energy balance of the whole body through dissipating large amounts of chemical/food energy as heat 1. BAT&#8217;s most unique characteristics include plentiful uncoupling protein-1 (UCP-1) expression, abundant small oil droplets, a large number of mitochondria in a single cell, and significant vascularization in the tissue 2-5. These unique features strongly associate with the tissue&#8217;s important role in metabolism and energy expenditure. BAT was previously considered to be no longer present and possessing no significant physiological functions in adult humans 1, however recent PET/CT imaging investigations have clearly demonstrated that BAT still presents in human adults 2,3,6-9. An inverse correlation between BAT mass and body mass index (BMI) was established from several studies, and recent studies indicated that physical exercises could increase the mass of BAT. These results strongly suggest that the dysfunction of BAT is tightly associated with the pathologies of obesity and diabetes 2,6,10,11. In addition, mounting evidence indicates that the function of BAT is strongly related to various other pathologies such as neurodegenerative disease and cancer 3,7,12,13.
BAT activation, a process to increase thermogenesis, can be achieved under various conditions such as cold exposure, exercise, and drug treatment and gene manipulation 1,14,15. Cold exposure and norepinephrine treatment are the most used methods to activate BAT. Cold, which can be sensed by various mechanisms such as thermoreceptors in the skin, stimulates sympathetic nerves and leads to the release of norepinephrine (NE) to BAT. The released NE triggers UCP-1 to initialize thermogenesis to maintain the normal body temperature. Under this condition, the uptake of glucose also increases to provide more carbon sources for the increased metabolism in BAT 1,16,17. PET imaging with 18F-FDG has confirmed that the uptake of labeled glucose increased under cold conditions in human studies 6.
In terms of optical imaging, BAT is an ideal target. The interscapular BAT has a unique location in mice, situated away from larger organs such as the liver, heart, and stomach. Therefore, signal interference from these large organs is insignificant (Figure 1a). Meanwhile, the shallow location of interscapular BAT allows more signals to be captured by the detection camera. Moreover, BAT is a concentrated mass organ, which confines the light signal in certain areas. Additionally, the unique triangular physical shape of BAT makes it easy to distinguish from other tissues (Figure 1a).
Cerenkov luminescence imaging (CLI), a newly emerged molecular imaging technology 18-26, harnesses the luminescence generated from the + and - decay of radionuclides such as 18F and 131I in the medium. The charged particle (such as + and -) polarizes molecules while it travels in the medium 18-20, and luminescence/light is emitted when the polarized molecules relax back to equilibrium. The emitted luminescence is called Cerenkov Luminescence (CL). The unique spectral properties of CL include its broad spectrum throughout the ultraviolet (UV) and visible spectrum 18-20, and its inverse correlation between the intensity and the square of the wavelength (&#955;2). Both UV and visible ranges of the emitted light can be utilized for different applications. The UV portion of Cerenkov luminescence has been applied for in vivo photoactivation of caged luciferin 21, while the light emitted in the longer wavelength can be used for in vivo optical imaging 18,27-31.
For small animal studies, CLI imaging with an optical imaging system is faster and more cost effective than PET. Moreover, CLI can be applied for high throughput screening with an imaging system equipped with high throughput capacity. The advantages and disadvantages of this technology have been discussed in several reviews 25,32,33. 3D tomography of CLI has been intensively studied in several groups 28,34-37, and the applications of CLI for endoscopic imaging and intraoperative imaging have been successfully demonstrated in mice as well 30,38. In addition, Spinelli and Thorek et al. have demonstrated that CLI imaging could be applied to human subjects, thus the technology also has potential for clinical applications 39,40.
In the course of rediscovery of BAT in humans, PET images clearly indicated that a significant amount of 18F-FDG accumulated in BAT under certain conditions 2,3,6. In addition, PET imaging with mice also undoubtedly showed that BAT could be highlighted with 18F-FDG 4142. In this report, we demonstrate how Cerenkov luminescence emitted from 18F-FDG can be utilized for imaging BAT in small animals using an optical imaging system. Our approach provides a fast, cheap and convenient method of BAT imaging for small animals. This technique can be used as an alternative method for PET imaging with 18F-FDG, especially for laboratories without PET facilities.

<table><tbody><tr><td>Optical Imaging system</td><td>Perkin Elmer</td><td></td><td>IVIS Spectrum is an optical imaging system that is equiped with very sensitive camera for Cerenkov Luminescence. Some instrumental information is listed below: CCD Sensor: Back thinned, back illuminated&lt;br/&gt; CCD Size: 2.7 cm x 2.7 cm&lt;br/&gt; Pixels: 2048 x 2048&lt;br/&gt; Quantum Efficiency: 85%&lt;br/&gt; Min detectable photons: 70 photons/s/sr/cm&lt;sup&gt; 2 &lt;/sup&gt;&lt;br/&gt; Dark Current: &amp;lt;100 electrons/s/cm&lt;sup&gt; 2 &lt;/sup &gt;&lt;br/&gt; Lens: f 0.95 50 mm&lt;br/&gt; CCD Cooling: Cooled to 90 degree.&amp;nbsp;</td></tr><tr><td>&lt;sup&gt;18&lt;/sup&gt;F-FDG</td><td>IBA Molecular</td><td></td><td></td></tr><tr><td>norepinephrine</td><td>Sigma</td><td>N5785-250MG</td><td></td></tr><tr><td>Ketamine/Xylazine</td><td>Sigma</td><td>K113-10ML</td><td></td></tr></tbody></table>

cerenkov luminescence imaging of interscapular brown adipose tissue

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

In Figure 1, interscapular BAT (Figure 1a) was highlighted at all time points (30, 60, 120 min), and the contrast between BAT and the reference area can be easily observed (Figure 1b). Notably, the contour of the BAT image closely reflected its physical appearance, which has a triangular contour. The signal ratios between BAT and the reference area were 2.37, 2.49, and 2.53 fold at 30, 60, and 120 min after injection (Figure 1a).
From the step-wise dissection experiment, 85% of the signal at the interscapular site had originated from the BAT (Figure 2). However, there was still some residual signal located near the upper rim of the BAT, which probably originated from the BAT residual tissue and other adjacent tissues that include blood vessels and muscles.
It has been reported that BAT activation can be accomplished through Norepinephrine (NE) treatment and cold exposure 3,6. First, we observed BAT activation with NE in the same group of mice with and without NE treatment under short (5 min) isoflurane anesthesia. The data showed significantly higher CLI signal under the NE-treated condition (1.23 fold) than without NE treatment (Figure 3a).
In human studies, PET imaging showed that subjects under cold exposure had much higher 18F-FDG uptake in the BAT than those at room temperature 6. In this mice study, a 39% increase in 18F-FDG uptake was observed in the BAT of the animals treated with cold exposure (Figure 3b).
In mice, BAT activity can be influenced by different anesthesia regimens 3,41. For example, 18F-FDG uptake in BAT can be considerably decreased after one hour of anesthesia with ketamine 41. Comparing the 18F-FDG uptake in the same group of mice under short (5 min with isoflurane) and long anesthesia (70 min with ketamine/xylazine) regimens, a 54% decrease in 18F-FDG BAT uptake was observed in the group anesthetized with ketamine/xylazine (Figure 3c).
It is well known that heme-containing proteins (such as hemoglobin in blood and cytochrome c in mitochondria) can lead to significant light absorption, and it is expected that the abundant blood vessels in BAT 1 will change the spectrum of CLI, and the spectral shape from the BAT area will be different from other areas. Conceivably, spectral unmixing techniques allow us to separate the two CLI spectra. From Figure 4a, no particular area was highlighted from the unmixed image of component #1 (Unmix #1), and the corresponding spectrum (blue line in Figure 4d) closely resembled the emission spectrum of 18F in pure media, in which the CLI intensity is inversely correlated with the square of the wavelength 18,20. This data suggests that Unmix #1 represented an unspecific CLI signal, which is probably from very shallow depths, such as 18F-FDG accumulation in the skin. The peak of the unmixed #2 spectrum (red line) was around 640 nm, suggesting the signal was from the BAT. Interestingly, contrary to the significant attenuation of the intensity of CLI shorter than 640 nm, no dramatic decrease of intensity was observed once it reached its peak (red line in Figure 4), indicating better tissue penetration of the emitted light at longer wavelengths.
Multispectral Cerenkov luminescence tomography (msCLT) was used for the 3D reconstruction. msCLT has been previously reported by Kuo et al. 28,43, and is constructed from a set of 2D planar images acquired with a number of narrow bandpass filters (&#62;5 filters). The reconstructed 3D images are coregistered with surface tomography that is generated from the structure light, and the images reveal that a substantial portion of the CLI signal originated from interscapular BAT (Figure 5). Remarkably, the coronal image (Figure 5a) clearly outlines the two lobes of BAT, which closely resemble the triangle contour of the BAT shown in Figure 5e.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51790/51790fig1highres.jpg" width="500" /> 
Figure 1: (a) Triangular contour of interscapular BAT (blue arrow) in a mouse; (left) BAT is covered with white adipose tissue, and (right) BAT is exposed. (b) CLI images of a mouse at 30 and 60 min after 18F-FDG (280 &#181;Ci) intravenous injection. The images clearly outline the contour of BAT. (c) Quantitative analysis of CLI signal from interscapular BAT area and a reference area. Reprint from reference 42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51790/51790fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51790/51790fig2highres.jpg" width="500" /> 
Figure 2: (a) Representative images of mice before (left) and after (right) BAT removal. (b) Quantification indicates that &#62;85% CLI originated from BAT. Reprint from reference 42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51790/51790fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51790/51790fig3highres.jpg" width="500" /> 
Figure 3: (a) Representative CLI images of BAT of mice with (left) and without (right) NE stimulation under short isoflurane anesthesia. (b) Quantitative analysis of the CLI signals from the two groups in (a) (n = 4 for each group). (c) Representative CLI images of BAT of mice with (left) and without (right) cold stimulation under short isoflurane anesthesia. (d) Quantitative analysis of the CLI signals from the two groups shown in (e) (n = 3 - 4 for each group). (e) Representative CLI images of BAT at 60 min after 18F-FDG injection under short isoflurane anesthesia (5 min) (left) and ketamine/xylazine anesthesia (70 min) (right). (f) Quantitative analysis of the CLI signals from the two groups shown in (g) (n = 4 for each group). Reprint from reference 42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51790/51790fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51790/51790fig4highres.jpg" width="500" /> 
Figure 4: Spectral unmixing for unspecific CLI and BAT CLI. (a) Unmix #1 shows that unspecific CLI signal from 18F-FDG is distributed over the whole body (the unmixed spectrum for this image is shown in (d) (blue line)). (b) Unmix #2 indicates that the majority of CLI at the interscapular site is from BAT (the unmixed spectrum is shown in (d) (red line)). The CLI peak is around 640 nm. (c) Merged image of Unmix #1 and #2. (d) The CLI spectra of Unmix #1 and #2. Reprint from reference 42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51790/51790fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51790/51790fig5highres.jpg" width="500" /> 
Figure 5: Multispectral Cerenkov luminescence tomography. (a - d) 3D reconstruction of the images. Interscapular BAT can be seen in coronal (a), sagittal (b), and transverse (c) views, as well as in the 3D image (d); (e) Physical BAT shape is shown (blue arrow) which correlates with reconstructed images. Reprint and adapt from reference 42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51790/51790fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue at JoVE.com

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

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

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

cerenkov-luminescence-imaging-of-interscapular-brown-adipose-tissue

Nanyang Technological University

Research

JoVE Journal

Medicine

13.3K Views.  Massachusetts General Hospital/Harvard Medical School. In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Video: Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

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

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

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

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

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

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

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

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

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

Biology

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, typically have narrow absorbances, such that the mean free path of light outside the absorbance peaks can be quite long. As people cannot see through tissue, they generally imagine that tissues like the brain, fat, and bone contain little or no light. However, photoresponsive opsin proteins are expressed within many of these tissues, and their functions are poorly understood. Radiance internal to tissue is also important for understanding photosynthesis. For example, giant clams are strongly absorbing yet maintain a dense population of algae deep in the tissue. Light propagation through systems like sediments and biofilms can be complex, and these communities can be major contributors to ecosystem productivity. Therefore, a method for constructing optical micro-probes for measuring scalar irradiance (photon flux intersecting a point) and downwelling irradiance (photon flux crossing a plane perpendicularly) to better understand these phenomena inside living tissue has been developed. This technique is also tractable in field laboratories. These micro-probes are made from heat-pulled optical fibers that are then secured in pulled glass pipettes. To change the angular acceptance of the probe, a 10-100 &#181;m sized sphere of UV-curable epoxy mixed with titanium dioxide is then secured to the end of a pulled, trimmed fiber. The probe is inserted into living tissue, and its position is controlled using a micromanipulator. These probes are capable of measuring in situ tissue radiance at spatial resolutions of 10-100 &#181;m or on the scale of single cells. These probes were used to characterize the light reaching the adipose and brain cells 4 mm below the skin of a living mouse and to characterize the light reaching similar depths within living algae-rich giant clam tissue.

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

Organisms appear opaque largely because their outer tissue layers are strongly scattering to incident light; strongly absorbing pigments, such as blood, ...

Bioengineering

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

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

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

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

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

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...

Immunology and Infection

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 ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

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

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

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

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

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

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

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