1.

Introduction

Sentinel lymph node (SLN) biopsy has become the standard of care for patients diagnosed with breast cancer, replacing axillary lymph node dissection. Biopsy of the lymph nodes directly draining the tumor area can accurately stage cancer to guide therapeutic decisions. Ultrasound (US) guidance of minimally invasive interventions is widely performed, leading to improved treatment outcomes and shorter recovery.^{1, 2} Real-time, noninvasive, nonionizing, and portable US imaging is currently the primary method for guiding targeted needle biopsies and nerve blockings.^{3, 4}

Photoacoustic (PA) imaging is a nonionizing and noninvasive hybrid imaging technique that combines strong optical absorption contrast with high ultrasonic spatial resolution.⁵ Since ultrasonic scattering is 2 to 3 orders of magnitude less than optical scattering, PA imaging can break through the fundamental depth limitations of pure optical imaging.⁶ Using intrinsic contrasts such as hemoglobin or melanin, PA imaging can provide both morphological and functional information.⁵ Moreover, using molecularly targeted exogenous contrast agents, it can also provide molecular information.⁵ We previously reported in vivo detection of lymph nodes in rats using a raster-scanning tabletop PA imaging system. Methylene blue,⁷ indocyanine green (ICG),⁸ and gold nanoparticles⁹ were used as contrast agents. However, this system was relatively slow for clinical applications ( $20\min$ for a $20\text{-}\times 20\text{-}{\mathrm{mm}}^2$ field of view), and its imaging head was not handheld. We also reported in vivo PA and US mapping of SLNs in rats using a US array, but the light delivery was not handheld.¹⁰

In this paper, for the first time to our knowledge, we report noninvasive in vivo mapping of deeply positioned ICG-dyed SLNs $(\sim 2.1\mathrm{cm})$ in rats using a PA imaging system adapted from a commercial US array system. A US probe integrated with optical fiber bundles for light delivery enabled handheld scanning as in ultrasonagraphy.^{11, 12} ICG uptake in SLNs was confirmed by fluorescence imaging. Furthermore, both real-time PA imaging [1 frame per second (fps)] and US imaging $\left(5\mathrm{fps}\right)$ successfully guided needle insertion in vivo.

2.

Methods and Materials

Figure 1 shows an experimental schematic of the integrated PA and US imaging system, adapted from a clinical US imaging system¹⁰ (iU22, Philips Healthcare). Raw per-channel PA and US data were both acquired through the modified channel board architecture and transferred to a custom-made data acquisition computer for display and postprocessing. The data acquisition system was synchronized with laser firing. Fourier-beam-formed PA images were displayed at a frame rate of $\sim 1\mathrm{fps}$ . However, the frame rate can be improved to $10\mathrm{fps}$ , limited by the current laser repetition rate. US images were captured at $5\mathrm{fps}$ . For this study, we used a linear array US probe (L8-4, Philips Healthcare) with a nominal bandwidth of $4\text{to}8\mathrm{MHz}$ . Light from a tunable dye laser (NS, Sirah), pumped by a $Q$ -switched Nd:YAG laser (PRO-350-10, Newport) with a $6.5\text{-}\mathrm{ns}$ pulse duration and a $10\text{-}\mathrm{Hz}$ pulse repetition rate, was coupled to a multimode fiber bundle (CB18043, Fiberguide). The fiber bundle was bifurcated and physically integrated with the US probe, enabling handheld scanning. The two optical beams were obliquely incident $(\sim 45\mathrm{deg})$ on the tissue surface as rectangles ( $2\times 3\mathrm{cm}$ along the $X$ and $Y$ axes from each bundle). An optical wavelength of $810\mathrm{nm}$ , close to the peak optical absorption wavelength of ICG ( $806\mathrm{nm}$ in albumin), was utilized. Light fluence on the skin was less than $1.5\mathrm{mJ}∕{\mathrm{cm}}^2$ , well below the American National Standards Institute safety limits.¹³ Tabletop PA imaging systems and the previously reported study using this modified Philips system required a water tank, which is clinically undesirable. Here, US gel was applied to directly couple the PA probe to the surface of the tissue.

Fig. 1

Schematic of the integrated PA and US imaging system based on a clinical ultrasound scanner (iU22, Philips Healthcare).

Animal handling was performed according to the guidelines on the care and use of laboratory animals at Washington University in St. Louis. Sprague Dawley rats $(\sim 200\mathrm{g})$ were initially anesthetized using a mixture of ketamine $(85\mathrm{mg}∕\mathrm{kg})$ and xylazine $(15\mathrm{mg}∕\mathrm{kg})$ . To test the clinical feasibility of this technique on humans, we intentionally increased the imaging depth by laying $\sim 2\mathrm{cm}$ of chicken tissue atop the rat. After depilation in the left axillary region, we first obtained a control PA image before the injection of ICG. After intradermally injecting $0.1\mathrm{ml}$ of $1\text{-}\mathrm{mM}$ ICG into the left forepaw, we acquired a series of PA images to monitor the ICG uptake in the SLN dynamically. Then, we demonstrated PA image-guided needle $\left(18\text{gauge}\right)$ insertion in vivo. Here, we mechanically fixed the PA imaging probe to avoid motion artifacts. After PA imaging, in vivo fluorescence imaging was performed with a time-domain diffuse optical imaging system (eXplore Optix, Advance Research Technologies) to confirm ICG uptake. Then, ex vivo fluorescence imaging was performed to confirm uptake in the lymph nodes using a planar reflectance imaging system (Kodak IS4000MM, Carestream Health) with $755\text{-}\mathrm{nm}$ excitation and $830\text{-}\mathrm{nm}$ detection.

3.

Results and Discussion

To investigate the feasibility of PA and US mapping of SLNs with ICG, we imaged the left axillary region in a rat before and after ICG injection. A control PA B-scan image was acquired before injection [Fig. 2 ]. Immediately after injection, ICG accumulation in the deeply positioned SLN was observed photoacoustically. Video 1 shows the dynamic accumulation of ICG in the SLN. The PA image of the ICG-dyed SLN acquired at $10\min$ postinjection [Fig. 2] shows that ICG accumulation enhanced the PA signal in the SLN by $9.5\pm 2.7$ (standard deviation). Figure 2, created by overlaying the PA and US images, shows both morphological information and functional information (ICG uptake in the SLN). For comparison, the PA images in Figs. 2, 2, 2 are displayed in the same dynamic range. A snapshot of the US-guided needle insertion is shown in Fig. 2. As the needle was inserted into the chicken tissue in vivo, it was ultrasonically tracked in real time (Video 2 ), but the SLN was not detected ultrasonically. The needle progression could also be detected photoacoustically, as shown in Fig. 2 and Video 3 . PA imaging was able to detect the SLN clearly. Comparison of the two videos clearly shows that PA imaging has much higher contrast than US imaging in needle tracking under the current experimental condition. We quantified the image contrasts of the needles in both images within the regions of interest indicated by the yellow dotted boxes in Figs. 2 and 2. The PA image contrast of the needle was $8.5\pm 4$ (standard deviation), whereas the US image contrast was $1.2\pm 0.04$ (standard deviation). However, in neither case was the tracking of the needle optimized and both imaging modalities were highly operator-dependant.

Fig. 2

In vivo PA and US B-scan imaging of an SLN dyed with ICG and guiding of a biopsy needle: (a) control PA image acquired before ICG injection, (b) PA image taken $10\min$ after ICG injection, (c) overlaid PA (pseudocolor) and US (gray scale) images, (d) US guidance of a biopsy needle, and (e) PA guidance of a biopsy needle. The image contrasts were calculated using the values within the regions of interest indicated by the yellow dotted boxes in (d) and (e). PA, photoacoustic; US, ultrasound; ICG, indocyanine green; and SLN, sentinel lymph node. (Color online only.)

Video 1

Dynamic PA imaging of ICG accumulation in the SLN in vivo (QuickTime, 7.4 MB). .

Video 2

US guidance of a biopsy needle (QuickTime, 6.9 MB). .

Video 3

PA guidance of a biopsy needle (QuickTime, 6.6 MB).

Fluorescence images obtained after PA imaging confirmed ICG uptake in the SLN [Fig. 3 ]. Further, we imaged fluorescently three dissected lymph nodes (LNs) from the left axillary region [Fig. 3]. Only the first draining node, the sentinel node, generated strong fluorescence.

Fig. 3

Fluorescence imaging of an SLN dyed with ICG: (a) fluorescence image of the left axillary region showing high intensity from the SLN and lymph vessels, acquired noninvasively $\sim 1\mathrm{h}$ after injection of ICG and (b) ex vivo fluorescence image of LNs dissected from the same axillary region after in vivo imaging. Grayscale represents white light intensity, and pseudocolor represents fluorescence intensity. SLN, sentinel lymph node; ICG, indocyanine green; LN, lymph node; and FL, fluorescence.

4.

Conclusions

We developed a noninvasive, nonionizing, real-time, and handheld PA and US imaging system using a modified clinical US imaging system. In vivo mapping of rat SLNs at an imaging depth of $\sim 2.1\mathrm{cm}$ was successfully accomplished following ICG injection. Further, PA imaging enabled guided needle insertion with strong contrast in vivo. This technique is highly translatable to clinical applications for image-guided SLN biopsy, nerve blockings, and many others.