Name: a novel technique for generating and observing chemiluminescence in a biological setting
Uploaded: 2017-03-09T12:30:00
Description: Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

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
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	<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 ...

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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.

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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>
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			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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			<td style="width:121px;">Cut from a 3/4&#8221; x 24&#8221; x 30&#8221; birch plywood sheet</td>
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			<td style="width:119px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
			<td style="width:121px;">Comments</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="22">
			<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 ...

The overall goal of this protocol is to offer a new preclinical, interoperative, chemiluminescent imaging technique that can provide benefits over existing optical techniques in terms of signal-to-noise ratios and detections limits. This method can help answer key questions that have not been accessible by traditional optical imaging techniques in many fields related to the biological sciences. The main advantages of this technique are that it has a high signal-to-noise ratio and that it requires neither instant light nor radiation.So what we found is that the implications of this technique really extends towards interoperative imaging of multiple disease states including cancer because of the high signal-to-noise ratios that this technique has to offer. As incident light is not needed for this technique there are no issues with incident light absorption. Further, there is a large chemical tool box available to functionalize the chemiluminescent core molecule.To begin the procedure prepare 100 microliter standard solutions of tris(bypiridine)ruthenium(II)chloride in reverse osmosis water. Then, combine each standard with 100 microliters of a 25 millimolar solution of ammonium cerium nitrate in reverse osmosis water on a microscope slide. Open the bioluminescence reader imaging software and click initialize.In imaging mode, ensure that luminescent and photograph are checked and florescent is unchecked. Set the luminescent exposure time, binning, f/stop and emission filter parameters. Set the photograph exposure time, binning and f/stop parameters.Then set the subject height accordingly to the sample to be imaged. Set the field of view to B for a 14-centimeter distance between the camera and the sample stage. Cover the floor of the bioluminescence reader imaging chamber in the sample stage with black construction paper.Then place the first slide with the droplet of standard mixture on the sample stage with the droplet centered in the green lightbox crosshair. Next, fill the three-ounce spray bottle of a custom-built nebulizer with the 1:3 solution of triethylamine in a 50/50 solution of water and ethanol. Ensure that there are no air bubbles in the aspiration pipe.Place the nebulizer assembly in the imaging chamber with the spray nozzle pointed towards the droplet to be imaged without obstructing the camera view of the droplet. Ensure that the nebulizer power switch and power indicator are on, the magnetic toggle switch is off and the power nebulizer and remote cords are not tangled. Cover hot spots such as marks on the slide with small pieces of black construction paper.Place at least 40 centimeters of the nebulizer remote cord in the imaging chamber. Gently close the chamber door without crimping the nebulizer cords. In the instrument software, click acquire, enable autosaving and set the data file path.Initiate the imaging sequence. When the shutter opens with an audible click, switch the nebulizer toggle three times to spray three bursts of triethylamine solution onto the sample. Repeat the procedure with each standard slide to determine the minimum detectable concentration of the ruthenium imaging agent.To begin the in vivo imaging procedure, prepare 100 microliters of a solution containing between 8 and 33 nanomoles of tris(bypiridine)ruthenium(II)chloride in phosphate-buffered saline. Prepare a 25-millimolar aqueous solution of ammonium cerium nitrate. Next, intravenously inject 100 microliters of the ruthenium imaging agent into the tail vein of healthy mice.Sacrifice the mice 10 minutes after injection by carbon dioxide asphyxiation. Remove the skin from the torso with a Y cut and then remove the costal arch to expose the heart and lungs. Cut an outlet in the right atrium and inject 20 milliliters of PBS through a 24-gauge needle into the left ventricle.Cut through the belly skin to expose the kidney and liver. If the organ of interest is the liver, kidney or spleen make a visible longitudinal cut through the organ. Prepare a new imaging sequence in the bioluminescent reader software.For whole abdomen imaging, place the mouse in the imaging chamber with the open abdomen facing the camera and the head pointing towards the back of the instrument. Center the organ of interest in the green lightbox crosshair. Thoroughly wash out the spray bottle and nozzle from the nebulizer assembly and then fill the bottle with the ammonium cerium nitrate solution.Place the nebulizer in the imaging chamber with the spray nozzle directed at the imaging site. Ensure that the camera is unobstructed, the cords are not tangled and at least 40 centimeters of the remote cord are in the chamber. Close the chamber and start the imaging sequence.As the shutter opens with a click, switch the toggle three times to spray three bursts of the oxidizing agent onto the imaging site. After imaging, remove the nebulizer assembly and thoroughly rinse the spray nozzle to prevent crystallization of the ammonium cerium nitrate. For subsequent individual organ imaging and quantification, remove the mouse from the instrument and excise the inner organs starting from the open body cavity.Cut through the hind leg skin to excise muscle tissue samples. If the organ of interest is the liver, kidney or spleen cut the organ in half longitudinally. Then place the organ on a Petri dish or piece of black construction paper.Image each organ in the same way as the whole animal, rinsing the spray nozzle after each sequence. In this procedure, mice were injected with a ruthenium-based imaging agent and the tissue of interest sprayed with an oxidizer to induce chemiluminescence. The minimum detectable concentration of the imaging agent was determined to be 6.9 picomoles per centimeter squared in these conditions.Chemiluminescence in white light images of the organs of interest were obtained with a bioluminescence reader. The chemiluminescence signal was predominantly observed in the kidneys following intravenous injection indicating renal elimination of the hydrophilic tris(bypiridine)ruthenium(II)cation. The signal-to-noise ratio of the imaging agent versus PBS alone was 27:1 in the kidneys and 21:1 in the liver.Mouse popliteal lymph nodes were also evaluated with a subcutaneous injection of a more concentrated solution of the ruthenium-based imaging agent. The lymph nodes containing the imaging agent showed a significantly greater radiance than the lymph nodes containing PBS alone. Once mastered, this technique can be done in two hours if it is properly performed.While attempting this procedure, it's important to remember to exclude ambient light and to avoid contamination with ruthenium tris bpy. After its development, this technique will pave the way for researchers in the field of biomolecular imaging to explore disease states such as cancer in mice. Though this method can provide insight into questions that were not accessible by traditional optical imaging techniques, any question that could be investigated with traditional techniques potentially can be addressed with chemiluminescence.

Research

JoVE Journal

Bioengineering

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

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Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

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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 ...

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 ...

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

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...