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.