Fluorescence imaging is increasingly being employed for surgical guidance, used to minimize iatrogenic damage, expedite surgical procedures, and enhance surgical efficiency, with utility in various clinical fields.^{1, 2, 3} Applications of fluorescence image-guided surgery (FIGS) include sentinel lymph node mapping,^{2, 3} assessing coronary artery bypass graft patency,⁴ identifying tumors intraoperatively,^{5, 6} and minimizing the risk of iatrogenic bile duct injury.⁷ Many of these applications, such as imaging of lymphatics or vasculature, are enabled by nontargeted fluorescent contrast agents such as indocyanine green or methylene blue (MB).

Contrast agents emitting in the NIR region of the optical spectrum $\left(700\text{to}1000\mathrm{nm}\right)$ provide several advantages over agents in the visible region.⁸ Tissue constituents that dominate absorption of light in the visible have absorption minima in the red to NIR. Moreover, there is a dramatic decrease in scattering in the NIR relative to visible wavelengths. The reduced absorption and scattering results in less light attenuation and thus deeper penetration. Imaging in the NIR also minimizes background, as most endogenous fluorescent species emit in the visible spectrum.⁹ In FIGS, one channel typically images the fluorescence emission, while the other channel simultaneously captures a color video of the surgical area. Thus, emission in the NIR also allows for minimal spectral overlap between the color and fluorescence channels, resulting in higher color fidelity and higher fluorescence collection efficiency. FIGS instrumentation has been developed for both open and minimally invasive surgeries.^{2, 3, 4, 5, 6, 7} Several of these instruments feature only a single (fluorescence) channel,^{2, 4, 6, 7} or have a relatively large footprint.^{2, 3, 4} To improve access and increase the clinical utility of FIGS, particularly for open surgical applications,³ the size of existing imaging systems has to be reduced for greater flexibility and mobility. Here we describe the development of a compact dual-mode imaging probe and demonstrate its use in cardiovascular surgery in a preclinical model.

Our strategy for size reduction addresses illumination, detection, and optics. First, the system uses fiber optic delivery of both excitation and illumination. Conventional systems often house the light source, potentially including electronics and active cooling, in the image head.^{2, 6, 10} By displacing the light sources from the imaging head to a medical cart, we have eliminated the weight of the sources and thermal management. Second, we have replaced the imaging cameras with compact “lipstick” cameras. Prevalent in consumer electronics and security applications, these cameras are affordable, lightweight ( $\sim 25\mathrm{g}$ ,) and readily available for integration. In comparison to the substantially larger, high gain (e.g., intensified²) or actively cooled, scientific grade³ cameras often used in FIGS, consumer-grade lipstick cameras are less sensitive. For example, lipstick cameras have smaller active detector areas and/or smaller pixels, resulting in lower sensitivity. Thus, our third strategy to maintain compactness without compromising sensitivity was a custom optical design with high collection efficiency.

The compact instrument [Fig. 1 ] was designed for use with MB, a dye with a wide range of clinical utility approved by the United States Food and Drug Administration. While it is commonly used as a stain with deep blue hue, MB is also a fluorophore in the NIR, emitting around $700\mathrm{nm}$ .^{3, 11} Excitation light was provided by a monochromatic $500\text{-}\mathrm{mW}$ (class 3b), $660\text{-}\mathrm{nm}$ laser diode (PLT Technology PLT-660, Santa Barbara, California) delivered to the imaging head through an excitation clean-up filter (Semrock FF01-655/40, Rochester, New York) and a $2\text{-}\mathrm{mm}$ plastic fiber (Timbercon CA-005-255-255-001M-197, Lake Oswego, Oregon). Approximately $300\mathrm{mW}$ of excitation power emerged from the fiber distal end, corresponding to $\sim 60\mathrm{\%}$ coupling efficiency. A $4.6\text{-}\mathrm{W}$ white LED (Vision Light Tech HSL-58SW-D300, Uden, The Netherlands) provided white light illumination through a second delivery fiber. A $15\text{-}\mathrm{mm}$ focal length aspherical lens (Thorlabs AL1815-A, Newton, New Jersey) was used to generate a top-hat uniform illumination at a working distance of $75\text{to}300\mathrm{mm}$ . Fluorescence emission from the sample was collected through a dichroic mirror (Semrock FF677-Di01), an emission filter (Semrock FF01-716/40,) and a custom designed and fabricated fixed-focus Cooke triplet (Table 1 ), designed to operate from $450\text{to}1000\mathrm{nm}$ . The entrance pupil diameter (EPD) of this lens is large $(\sim 7\mathrm{mm})$ compared to the off-the-shelf optics ( $\sim 3\mathrm{mm}$ , Watec Cameras, Orangeburg, New York) used on the white light channel, translating to roughly five times higher collection efficiency. The white light (color) and fluorescence (monochrome) channels each have a dedicated, lipstick charge-coupled device camera (Watec 240 and 704R, respectively). The analog outputs of both cameras were digitized at 30 frames per second using a dual channel framegrabber (Matrox Morphis MOR/2VD, Dorval, Quebec) and displayed using custom image acquisition software developed in C++ and Matrox MIL-8.0. Video streams from both channels were recorded uncompressed in AVI format to a RAID disk array in real time. Collectively, the imaging head weighed less than $0.5\mathrm{kg}$ , with a total volume of approximately $65\times 40\times 40{\mathrm{mm}}^3$ . The total instrument cost was $\sim \mathrm{\$}\mathrm{15,000}$ , roughly the cost of scientific-grade charge-coupled device (CCD)³ or intensified cameras² used in other FIGS instruments.

Fig. 1

(a) Schematic diagram and picture (inset) of compact FIGS instrument. (b) Performance of the FIGS hand-held system as a function of working distance. DOF is depth of field, and FFOV is the full field of view.

Table 1

Detailed design parameters of a custom Cooke triplet, designed with image side f∕number=1.8 , effective focal length=14.5mm , full field of view=16deg , and distortion <1% . R is radius, T is thickness, and Inf is infinity.

Theoretical performance parameters of the hand-held probe are shown as a function of working distance (WD) in Fig. 1. Irradiance at the sample drops geometrically with distance, while the illumination spot diameter, depth of field (DOF), and imaging full field of view (FFOV) increase linearly with WD. Using the laser diode excitation source, the irradiance at the sample can provide adequate excitation power density $(>5\mathrm{mW}∕{\mathrm{cm}}^2)$ at up to $\sim 300\mathrm{mm}$ WD. Optimized for a typical WD of $150\mathrm{mm}$ , the DOF is $\sim 15\mathrm{mm}$ , defined by the location where the modulation transfer function (MTF) drops by 15% relative to best focus. While $15\mathrm{mm}$ seems somewhat limited, a low DOF is a consequence of increasing the EPD. Furthermore, the image quality can be acceptable over a wider range, as a 15% drop in the MTF is quite stringent and maintains images with fairly high image quality (Fig. 2 ). An MTF of $>0.6$ is achieved at 50 line pairs per mm (lp/mm) at the image plane, corresponding to a sharp image quality and a high spatial resolution of $5\mathrm{lp}∕\mathrm{mm}$ $\left(100\mu \mathrm{m}\right)$ at the object plane. Sensitivity of the fluorescence channel is also shown in Fig. 2 by collecting images of $1\text{-}\mu \mathrm{M}$ MB in a $1.5\text{-}\mathrm{mm}$ -diam glass tube as a function of depth in 1% Intralipid. The geometry of the sample is consistent with that of a tubular structure such as the ureter, and the concentration of the dye is acceptable for clinical use.³

Fig. 2

Top: through focus white light images of a USAF 1951 resolution target, optimized at a $150\text{-}\mathrm{mm}$ working distance. The arrow corresponds to $3.56\mathrm{lp}∕\mathrm{mm}$ . Bottom: images of $1\text{-}\mu \mathrm{M}$ methylene blue fluorescence from a $1.5\text{-}\mathrm{mm}$ diameter tube under increasing depths of 1% Intralipid.

Following design and construction of the hand-held imaging device, we performed preclinical FIGS for an application in cardiovascular stem cell therapy. Implantation of stem cells to regenerate cardiomyocytes is an emerging technique for restoring heart function. Differentiation of stem cells into viable cardiomyocytes may vary depending on the injection site, preferably within the infarct or peri-infarct zone adjacent to healthy myocardium.¹² Thus a precise way to visualize this border zone along with healthy vasculature, i.e., tissue premapping, may be critical for efficacy of cardiovascular stem cell therapy.

Animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee at GE Global Research. We applied a well-established murine model of myocardial infarction that has been employed to examine the efficacy of therapeutic interventions on cardiac remodeling and survival.¹³ Briefly, the left anterior descending coronary artery in a male Wistar Hannover rat was ligated to induce an ischemic infarct in the free wall of the left ventricle. Over time, the infarct area is replaced by fibrotic tissue, and adjacent cardiac tissue becomes hypertrophic. Five months later, a second operation was performed to identify the viable regions of the heart, free of ischemia and fibrosis. The compact probe was mounted over the surgical field for hands-free operation at a working distance of $\sim 150\mathrm{mm}$ . A $50\text{-}\mu \mathrm{L}$ bolus of MB with $70\text{-}\mu \mathrm{M}$ concentration was injected into the left ventricle to highlight the vasculature in the healthy tissue. Whereas Fig. 2 demonstrated the high sensitivity of the imaging system, a higher dose was used for in vivo imaging, to increase signal levels while avoiding an adverse response to MB. The injected bolus dose of $70\mu \mathrm{M}$ (corresponding to $\sim 4\mu \mathrm{g}∕\mathrm{kg}$ ) was $\sim 500$ -fold lower than previous pharmacokinetics studies of MB.¹⁴

Due to the circulation of the fluorophore in the vasculature and its uptake by healthy tissue, fluorescence serves to provide negative contrast to the scar region, which was not visible under white light. The still images (Fig. 3 ) extracted from real-time $\left(30\mathrm{fps}\right)$ recorded video demonstrate high color fidelity and sharp focus. The addition of a fluorescence modality improves real-time intraoperative visualization of critical structures, in this case the precise location of potentially ischemic tissue resulting from the infarct. Additionally, the compact image head design allowed for rapid and flexible repositioning during surgery.

Fig. 3

Preclinical image-guided surgery for vasculature mapping in a rat heart at time of injection (top) and shortly $(\sim 60\mathrm{s})$ after injection (bottom). The fluorescence channel clearly indicates ischemic regions of the heart, approximated by the circle, which are otherwise inconspicuous in the white light channel. The field of view is approximately $25\times 25{\mathrm{mm}}^2$ .

In summary, we have developed a compact instrument for FIGS and demonstrated its use in a preclinical model. The system uses fiber optic delivery of laser diode excitation, along with a custom optical design with high collection efficiency and compact consumer-grade cameras, to minimize the volume and weight. While the current hand-held probe is designed for use with MB, it can easily be adapted for use with other contrast agents simply by replacing the excitation light source and filters. Dramatic size reduction increases flexibility and access, and allows for portable use or unobtrusive mounting over the surgical field. This instrument is complementary to other small form factor devices developed primarily for spectroscopic and tomographic applications.¹⁵