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

Summary

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.

Abstract

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.

Introduction

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't fit: the operating room. While whole body imaging techniq....

Protocol

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

1. 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 × 2.5 × 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 cm

Results

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.......

Discussion

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, technologies^4,5,6,7,8,9, this chemiluminescent reporter system employs an imaging probe that is non-radioactive and facilitates detect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wood part A (12.5 × 2.5 × 1.8 cm)	Woodcraft	131404	Cut from a 3/4” x 24” x 30” birch plywood sheet
Wood part B (12.7 × 10.7 × 1.8 cm)	Woodcraft	131404	Cut from a 3/4” x 24” x 30” birch plywood sheet
Wood part C (11 × 2.5 × 1.8 cm)	Woodcraft	131404	Cut from a 3/4” x 24” x 30” birch plywood sheet
Screws (4 × 25 mm)	Screwfix	79939
Harmon Face Values 3 oz mini sprayer	Bed, Bath and Beyond
stainless steel rod (10 cm of 1/16” steel)	Metals Depot Int. Inc.	2192
Pencil Classic HB	Papermate	58592
Paper clip	Office Depot	221720
speaker cable	RCA Inc.	AH1650SN
Energizer 9V alkaline battery	Energizer Holdings Inc.	EN22
Hitech HS-82MG Micro Servo Motor, 3.4 kg/cm output torque @ 6V	Hitech RCD USA Inc.	32082S
Name	Company	Catalog Number	Comments
28 cm plastic cable ties	General Electric Inc.	50725
Duct tape	3M Inc.	3939
littleBits w1 wire	littleBits Inc.	w1 wire
littleBits p1 power	littleBits Inc.	p1 power
littleBits i2 toggle switch	littleBits Inc.	i2 toggle switch
littleBits 011 servo	littleBits Inc.	011 servo
20 cm plastic covered wire twist ties	Four Star Plastics	71TIE8000
Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate	Sigma-Aldrich Inc.	224758
Ammonium cerium(IV) nitrate	Sigma-Aldrich Inc.	22249
Isofluorane	Baxter Healthcare	1001936060
PBS	Sigma-Aldrich	PBS1
Ethanol	Sigma-Aldrich	2854
Triethylamine	Sigma-Aldrich Inc.	T0886
Water			Water was purified using a Milipore Mili-Q (R ≥ 18 MΩ)
Female nude (outbred) mice	Jackson Laboratories	1929	age 5 - 6 weeks
Strain C57BL/6J
NU/J male mice at	Jackson Laboratories	2019	age 6 – 8 weeks
IVIS 200 bioluminescence reader	Caliper Live Science
Live Image 4.2 software	Perkin-Elmer	128165
Microscope slides	ThermoScientific	4951PLUS4