The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

Ethics statement: All of the in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Memorial Sloan Kettering Cancer Center (MSK) Institutional Animal Care and Use Committee (IACUC).
1. Construction of a Nebulizing Device
<ol>
	<li>Attach wood part A (12.5 x 2.5 x 1.8 cm3) upright in the center of part B (12.7 x 10.7 x 1.8 cm3) using two screws (4 x 25 mm2). Attach wood part C (11 &#215; 2.5 &#215; 1.8 cm3) to the middle of part A (12.5 x 2.5 x 1.8 cm3) using one screw, such that part C (11 x 2.5 x 1.8 cm3) can still be moved. See Figure 1.</li>
	<li>Drill two holes through the lower tip of the spray bottle trigger of a plastic 3 oz mini sprayer (D) and push a stainless steel rod (10 cm of 1/16&#34; steel) (E) through to form two loops, one on either side of the trigger.</li>
	<li>Wrap the bottom part of the spray bottle with duct tape (F) to prevent the cable ties from slipping off. Attach the spray bottle to wood part C (11 x 2.5 x 1.8 cm3) using the two plastic cable ties (28 cm) (G).</li>
	<li>Cut off the 011 servo motor (I) and reconnect it with the loose cables of the servo control (H). Then, attach the servo motor to the top of wood part A (12.5 x 2.5 x 1.8 cm3) using duct tape.</li>
	<li>Attach a pencil (J) to the servo motor lever using the paper clip (K). Tightly connect the outermost parts of the pencil to the steel rod loops using plastic covered twist wires (L) and secure the ends on the pencil with duct tape.</li>
	<li>Cut off the servo motor control unit&#39;s magnetic cable connector (M) and reattach it to the speaker cable (N). Then, tape the servo motor control unit to wood part B (12.7 x 10.7 x 1.8 cm3). Cut a w1 cable with magnetic connectors in half and attach one part to the loose end of the copper speaker cable (1 m). Connect the (magnetic) i2 toggle switch and p1 power to the available w1 cable and a 9V battery.</li>
</ol>
2. Sensitivity Determination of the Method
<ol>
	<li>In a 1.5 mL microcentrifuge tube, prepare solutions of [Ru(bpy)3]Cl2 in reverse osmosis water (100 &#181;L) in amounts of 260 &#181;g (347 nmol), 52 &#181;g (69 nmol), 26 &#181;g (34 nmol), 5 &#181;g (6.9 nmol), 3 &#181;g (3.5 nmol), 520 ng (694 pmol), 260 ng (347 pmol), 52 ng (69 pmol), 26 ng (34 pmol), 5 ng (6.9 pmol), and 3 ng (3.5 pmol).</li>
	<li>Mix 100 &#181;L of each [Ru(bpy)3]Cl2 solution with 100 &#181;L of an aqueous solution of ammonium cerium nitrate ((NH4)2Ce(NO3)6) in water (25 mM) on a microscope slide.</li>
	<li>Set up the acquisition in the bioluminescence reader by initializing the imaging software.
	<ol>
		<li>Sign in to the user profile and look for the acquisition control panel. Click on &#34;Initialize&#34; and wait until the instrument is ready.</li>
		<li>Look for &#34;Imaging Mode&#34; and make sure &#34;Luminescent&#34; and &#34;Photograph&#34; are checked and that &#34;Fluorescent&#34; is unchecked.</li>
		<li>Change &#34;Exposure Time&#34; setting for &#34;Luminescent&#34; to 20 s. Set the remaining settings for &#34;Luminescent&#34; as follows: &#34;Binning&#34;: Medium; &#34;F/stop&#34;: 1; and &#34;Emission Filter&#34;: Open. 
		NOTE: The exposure times might need to be adapted according to the instrumentation and experimental setting used, if different from the presented setup.</li>
		<li>For &#34;Photograph,&#34; use the following settings: &#34;Exposure Time&#34;: Auto; &#34;Binning&#34;: Medium; and &#34;F/stop&#34;: 8. Adjust the &#34;Subject Height&#34; according to imaging target.Look for the &#34;Field of View&#34; dropdown menu. The initial setting is &#34;C.&#34; Change to &#34;B&#34; (14 cm distance between the camera and sample stage).</li>
	</ol>
	</li>
	<li>Set up the nebulizer by placing a microscopic slide on a sheet of black construction paper on the floor of the imaging chamber to protect it from the oxidizing agent. Mix a 100 &#181;Ldroplet of [Ru(bpy)3]Cl2-solution with 100 &#181;L of an aqueous solution of (NH4)2Ce(NO3)6. Observe the green box crosshair.
	<ol>
		<li>Place the imaging subject on the black construction paper, such that the area of interest is in the center of the green light box crosshair displayed on the sample stage. Prepare the nebulizer by detaching the plastic spray bottle from the wooden support. Fill a solution of triethylamine (1:3 in water/ethanol) into its plastic reservoir and reattach it to the wooden support.</li>
		<li>Place the nebulizer inside the bioluminescence reader and make sure that the power cord is disconnected from the nebulizer cord. Ensure that the power switch is on, the toggle switch is off, and the red LED is lit. Place the nebulizer such that the spray flow is pointed towards the area of interest on the imaging subject, while minimizing the view obstruction from the camera towards the imaging subject by the spray nozzle head.</li>
		<li>Place small, black pieces of construction paper over any potential hot spots (e.g., white marks on microscopic slides or injection sites) to shield them from spray. Place at least 40 cm of the nebulizer remote cord inside the imaging chamber, such that it does not interfere with the imaging subject, the nebulizer, or the magnetic door latch. Close the imaging system door. 
		NOTE: The crosshair will change size based on the &#34;Field of View&#34; setting in &#34;Living Image;&#34; make sure that this is set to &#34;B&#34;.</li>
	</ol>
	</li>
	<li>Acquire an image by initiating the imaging sequence. Click &#34;Acquire&#34; in the &#34;Acquisition Control Panel.&#34; On the first imaging sequence, enable autosaving if desired (recommended) and choose a data folder. Ignore the &#34;Edit Imaging Labels&#34; dialog until the end of the sequence. 
	NOTE: The control software displays the instrument&#39;s actions step-by-step in real time. After preparing the measurement and moving the sample stage to the right position, it opens the camera shutter and counts the measurement time. The shutter opening can also be heard by a click sound generated by the machine.</li>
	<li>As the shutter opens, spray three bursts of a solution of triethylamine (1:3 in water/ethanol, 0.24 &#177; 0.04 mL per spray burst) by switching the toggle switch three times to generate chemiluminescence. 
	NOTE: The sample stage will move during the measurement. Leave enough (minimum 40 cm) cable inside the instrument to allow this. Make sure that the solution to be sprayed by the nebulizer can be aspired by the ascending pipe and that there are no air bubbles in the pipe. Have several spare batteries for the nebulizer ready in case needed.</li>
</ol>
3. In Vivo Imaging After Systemic Intravenous Injection
<ol>
	<li>In a 1.5 mL microcentrifuge tube, prepare 100 &#181;L of phosphate-buffered saline (PBS) solution containing between 8 and 33 nmol of [Ru(bpy)3]Cl2. Prepare an aqueous solution of (NH4)2Ce(NO3)6 in water (25 mM) at the same time.</li>
	<li>Intravenously inject 100 &#181;L of [Ru(bpy)3]Cl2 into the tail vein of healthy mice (n = 5).</li>
	<li>Euthanize the mice 10 min after injection via CO2 asphyxiation.
	<ol>
		<li>Remove the skin with a Y-cut from the torso, then remove the costal arch in a U-shape to expose the heart and lungs. Perfuse the mice by cutting an outlet in the right atrium and injecting 20 mL of PBS through a 24 gauge needle into the left ventricle18. Carefully cut through the belly skin and expose the kidney and liver. Cut longitudinally through the organs to create a visible cut.</li>
	</ol>
	</li>
	<li>Set up the acquisition as described in steps 2.3-2.6, with the following changes.
	<ol>
		<li>After thoroughly washing the nebulizer plastic reservoir, fill it with a solution of (NH4)2Ce(NO3)6 in water (25 mM) instead of triethylamine. 
		NOTE: It is important to thoroughly rinse the nebulizer nozzle after every use, since crystallizing (NH4)2Ce(NO3)6 may destroy the spray nozzle after several uses.</li>
	</ol>
	</li>
	<li>Use the whole animal or organ samples for imaging.
	<ol>
		<li>For whole abdomen imaging, position the mouse carcass with the open abdomen facing the camera and the head pointing to the back of the instrument. Center the organ to be imaged (e.g., liver or kidney) in the green light box crosshair.</li>
		<li>For individual organ imaging and quantification, remove the mouse from the imaging instrument and pin it down. Starting from the already-opened body cavity, excise the inner organs (e.g., kidney, liver, lung, muscle, spleen, brain, and heart). Cut through the hind leg skin to excise muscle tissue. Carefully open the skull with a scalpel to excise the brain.
		<ol>
			<li>If the organ of interest is liver, kidney or spleen, cut all organs in half longitudinally, place each organ on a petri dish or piece of black construction paper.</li>
		</ol>
		</li>
		<li>Follow the procedure described in steps 2.3-2.6 to establish the relative emission of the chemiluminescent tracer for single organs.</li>
	</ol>
	</li>
</ol>
4. In Vivo Imaging of Lymph Nodes
<ol>
	<li>Prepare 10 &#181;L of a PBS solution containing 80 nmol of [Ru(bpy)3]Cl2. Prepare an aqueous solution of (NH4)2Ce(NO3)6 in water (25 mM) at the same time.</li>
	<li>Inject 10 &#181;L of the solution subdermally into the hind paw of healthy mice (n = 5). As a negative control, inject the contralateral paw with 10 &#181;L of pure PBS. Sacrifice the mice via CO2 asphyxiation 15 min after the injection. Remove the skin on both hind legs to expose the lymph canals up to the popliteal lymph nodes.</li>
	<li>Set up the acquisition as described in step 3.4.</li>
	<li>Remove the popliteal lymph nodes from both hind legs, cut them in half, and spray them with oxidant on a Petri dish, as described before (step 3.5.3), for the purpose of quantification.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+for+Metals+in+Medicine:+How+Nanotechnology+May+Help+To+Shape+the+Future.">Challenges for Metals in Medicine: How Nanotechnology May Help To Shape the Future.</a> ACS Nano. 7, 5654-5659 (2013).</li><li>Hasan, K., Bansal, A. K., Samuel, I. D. W., Rold&#225;n-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Emission+of+Cationic+Iridium+(III)+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand.">Tuning the Emission of Cationic Iridium (III) Complexes Towards the Red Through Methoxy Substitution of the Cyclometalating Ligand.</a> Nat.Sci. Rep. 5, 1-15 (2015).</li><li>Truong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescence+detection+with+water-soluble+iridium(III)+complexes+containing+a+sulfonate-functionalised+ancillary+ligand.">Chemiluminescence detection with water-soluble iridium(III) complexes containing a sulfonate-functionalised ancillary ligand.</a> Analyst. 139 (22), 6028-6035 (2014).</li></ol>

The authors have nothing to disclose.

Here, we have presented a technology that is capable of optically delineating tissue via the emission of photons created by a chemiluminescent reporter. In contrast to other, more established, technologies4,5,6,7,8,9, this chemiluminescent reporter system employs an imaging probe that is non-radioactive and facilitates detect...

In recent decades, imaging technologies have revolutionized the way that physicians diagnose and monitor disease. These imaging technologies, however, have been largely limited to whole body imaging systems, such as positron emission tomography (PET), single photon-emission computed tomography (SPECT), computed tomography (CT), and magnetic resonance imaging (MRI). Particular attention has been paid to cancer, and technological imaging breakthroughs have greatly improved the way that this disease is diagnosed and treated. Despite these advances, there is one place where these imaging technologies just don&#39;t fit: the operating room. While whole body imaging techniques can help in surgical planning, they typically lack spatial resolutions high enough to help physicians determine in real-time whether all of the tumor tissue has been removed or residual tumor tissue remains hidden at the surgical margins1. Making sure that no infiltrative tumor margins are left behind is one of the most important surgical goals, and surgeons must walk a tight-rope between rigorous and cautious tissue resection. If too much is removed, unwanted side effects for the patient are exacerbated; if too little is removed, recurrence rates are increased2,3. Therefore, it is crucial to delineate accurate tumor margins, and we believe that chemiluminescent intraoperative imaging can help to improve the accuracy of the identification of tumor margins by helping surgeons to visualize malignant tissue that could otherwise remain undetected with established techniques.
There are many imaging technologies currently being investigated for their possible utility as intraoperative imaging systems. These include &#946;- and &#947;-radiation-emitting probes4, optical fluorescence5, Raman spectroscopy6,7, and Cherenkov luminescence8,9. To date, however, none of these have become established as standard clinical tools. Optical fluorescence imaging has so far proven to be the most promising of these techniques and is therefore the most explored. While it has already been shown to be a valuable tool for many applications, it is not without its limitations. Indeed, its principal drawback is the background fluorescence generated by inherently autofluorescent biological tissue. This background autofluorescent signal is a product of the excitation of the surrounding tissue, in addition to the fluorophore, by the external light source needed for the generation of a fluorescent signal. From a practical perspective, this autofluorescence can potentially lead to low signal-to-noise ratios, which can limit the utility of this technology in the operating room.
The principal advantage of chemiluminescence imaging over fluorescence imaging is that no excitation light is necessary. As a result, there is no background autofluorescence. In chemiluminescence imaging, the excitation energy is instead generated chemically. This process produces no unintended background signal and therefore can result in higher signal-to-noise ratios. This could ultimately result in the more precise and accurate detection of surgical margins. Somewhat surprisingly, the utility of this approach as an intraoperative imaging technique has remained unexplored10. Indeed, the closest example to this technique is the oxidation of luminol by myeloperoxidase in mice11,12,13. Chemiluminescent biomedical imaging is therefore a rather unexplored area of research that could offer the following advantages: (1) minimal autofluorescence resulting in a low background signal with higher signal-to-noise ratios; (2) tunable wavelengths of chemiluminescent emissions ranging from the visible to the near-infrared; and (3) functionalizable chemiluminescent complexes that, when combined with linker technologies and targeted biomolecules that already exist, provide access to whole libraries of targeted molecular imaging probes14.
This proof-of-principle study illustrates the potential utility of chemiluminescent imaging in the biomedical setting using a ruthenium-based imaging agent. The chemiluminescent properties of this compound are well studied, with investigations dating back to the mid-1960s15. Upon chemical activation, the agent produces light at around 600 nm16, which is well suited for medical imaging purposes. The activation energy is provided by a redox reaction that leads to an excited state-which has a lifetime of 650 ns in water17-followed by the generation of photons upon relaxation of this excited state. Through the use of a specially-designed remote nebulizer, we were able to detect the compound both ex vivo and in vivo. The results of initial experiments are very promising, suggesting further investigation of this technology.

<table border="0" cellpadding="0" cellspacing="0" width="542">
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			<td height="144" style="height:144px;width:215px;">Wood part A (12.5&#215;2.5&#215;1.8 cm)</td>
			<td style="width:119px;">&#160;Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Wood part B (12.7&#215;10.7&#215;1.8cm)</td>
			<td style="width:119px;">Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">Wood part C (11&#215;2.5&#215;1.8cm)</td>
			<td style="width:119px;">Woodcraft</td>
			<td style="width:87px;">131404</td>
			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Screws (4&#215;25 mm)</td>
			<td style="width:119px;">Screwfix</td>
			<td style="width:87px;">79939</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">Harmon Face Values 3oz mini sprayer</td>
			<td style="width:119px;">Bed, Bath and Beyond</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">stainless steel rod (10 cm of 1/16&#8221; steel)</td>
			<td style="width:119px;">Metals Depot Int. Inc.</td>
			<td style="width:87px;">2192</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Pencil Classic HB</td>
			<td style="width:119px;">Papermate</td>
			<td style="width:87px;">58592</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Paper clip</td>
			<td style="width:119px;">Office Depot</td>
			<td style="width:87px;">221720</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">speaker cable</td>
			<td style="width:119px;">RCA Inc.</td>
			<td style="width:87px;">AH1650SN</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Energizer 9V alkaline battery</td>
			<td style="width:119px;">Energizer Holdings Inc.</td>
			<td style="width:87px;">EN22</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:215px;">Hitech HS-82MG Micro Servo Motor, 3.4kg/cm output torque @ 6V</td>
			<td style="width:119px;">Hitech RCD USA Inc.</td>
			<td style="width:87px;">32082S</td>
			<td style="width:121px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Name</td>
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			<td style="width:121px;">Comments</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:215px;">28 cm plastic cable ties</td>
			<td style="width:119px;">General Electric Inc.</td>
			<td style="width:87px;">50725</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Duct tape</td>
			<td style="width:119px;">3M Inc.</td>
			<td style="width:87px;">3939</td>
			<td style="width:121px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits w1 wire</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">w1 wire</td>
			<td style="width:121px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits p1 power</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">p1 power</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">littleBits i2 toggle switch</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">i2 toggle switch</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">littleBits 011 servo</td>
			<td style="width:119px;">littleBits Inc.</td>
			<td style="width:87px;">011 servo</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">20 cm plastic covered wire twist ties</td>
			<td style="width:119px;">Four Star Plastics</td>
			<td style="width:87px;">71TIE8000</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="112">
			<td height="133" rowspan="2" style="height:133px;width:215px;">Tris(2,2&#8242;-bipyridyl)dichlororuthenium(II) hexahydrate</td>
			<td rowspan="2" style="width:119px;">Sigma-Aldrich Inc.</td>
			<td rowspan="2" style="width:87px;">224758</td>
			<td rowspan="2" style="width:121px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Ammonium cerium(IV) nitrate</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">22249</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">Isofluorane</td>
			<td style="width:119px;">Baxter Healthcare</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:121px;"></td>
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			<td height="22" style="height:22px;width:215px;">PBS</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">PBS1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Ethanol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:87px;">2854</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Triethylamine</td>
			<td style="width:119px;">Sigma-Aldrich Inc.</td>
			<td style="width:87px;">T0886</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:215px;">Water</td>
			<td style="width:119px;"></td>
			<td style="width:87px;"></td>
			<td style="width:121px;">Water was purified using a Milipore Mili-Q (R &#8805; 18 M&#937;)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Female nude (outbred) mice</td>
			<td rowspan="2" style="width:119px;">Jackson Laboratories</td>
			<td rowspan="2" style="width:87px;">1929</td>
			<td rowspan="2" style="width:121px;">age 5 - 6 weeks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Strain C57BL/6J&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:215px;">NU/J male mice at&#160;</td>
			<td style="width:119px;">Jackson Laboratories</td>
			<td style="width:87px;">2019</td>
			<td style="width:121px;">age 6 &#8211; 8 weeks</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:215px;">IVIS 200 bioluminescence reader</td>
			<td style="width:119px;">Caliper Live Science</td>
			<td style="width:87px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Live Image 4.2 software</td>
			<td style="width:119px;">Perkin-Elmer</td>
			<td style="width:87px;">128165</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Microscope slides</td>
			<td style="width:119px;">ThermoScientific</td>
			<td style="width:87px;">4951PLUS4</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

The nebulizer system described in protocol section 1 can be constructed from easily-available materials at a low cost. It is intended to be an inset for remote-triggered spraying of the reducing/oxidizing agent inside a bioluminescent reader (Figure 1). Our design allows for the safe operation of the nebulizer within the bioluminescence reader at a 14 cm distance from the lens. No fogging or blurring of the lens was observed during the operation. We selected the commercia...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

a-novel-technique-for-generating-observing-chemiluminescence

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.4K Views.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Video: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...