Todd N. Erpelding

Sentinel Lymph Nodes in the Rat: Noninvasive Photoacoustic and US Imaging with a Clinical US System

PURPOSE To evaluate in vivo sentinel lymph node (SLN) mapping by using photoacoustic and ultrasonographic (US) imaging with a modified clinical US imaging system. MATERIALS AND METHODS Animal protocols were approved by the Animal Studies Committee. Methylene blue dye accumulation in axillary lymph nodes of seven healthy Sprague-Dawley rats was imaged by using a photoacoustic imaging system adapted from a clinical US imaging system. To investigate clinical translation, the imaging depth was extended up to 2.5 cm by adding chicken or turkey breast on top of the rat skin surface. Three-dimensional photoacoustic images were acquired by mechanically scanning the US transducer and light delivery fiber bundle along the elevational direction. RESULTS Photoacoustic images of rat SLNs clearly help visualization of methylene blue accumulation, whereas coregistered photoacoustic/US images depict lymph node positions relative to surrounding anatomy. Twenty minutes following methylene blue injection, photoacoustic signals from SLN regions increased nearly 33-fold from baseline signals in preinjection images, and mean contrast between SLNs and background tissue was 76.0 +/- 23.7 (standard deviation). Methylene blue accumulation in SLNs was confirmed photoacoustically by using the optical absorption spectrum of the dye. Three-dimensional photoacoustic images demonstrate dynamic accumulation of methylene blue in SLNs after traveling through lymph vessels. CONCLUSION In vivo photoacoustic and US mapping of SLNs was successfully demonstrated with a modified clinical US scanner. These results raise confidence that photoacoustic and US imaging can be used clinically for accurate, noninvasive imaging of SLNs for axillary lymph node staging in breast cancer patients.

Deeply penetrating in vivo photoacoustic imaging using a clinical ultrasound array system

Using a hand-held photoacoustic probe integrated with a clinical ultrasound array system, we successfully imaged objects deeply positioned in biological tissues. The optical contrasts were enhanced by methylene blue with a concentration of ~30 mM. The penetration depth reached ~5.2 cm in chicken breast tissue by using 650-nm wavelength, which is ~4.7 times the 1/e optical penetration depth. This imaging depth was achieved using a laser fluence on the tissue surface of only 3 mJ/cm2, which is 1/7 of the American National Standards Institute (ANSI) safety limit (20 mJ/cm2). The noise equivalent sensitivity at this depth was ~11 mM. Further, after intradermal injection of methylene blue in a rat, a sentinel lymph node was easily detected in vivo, beneath a 2-cm thick layer of chicken breast. Also, blood located 3.5 cm deep in the rat was clearly imaged with intrinsic contrast. We have photoacoustically guided insertion of a needle into a rat sentinel lymph node with accumulated methylene blue. These results highlight the clinical potential of photoacoustic image-guided identification and needle biopsy of sentinel lymph nodes for axillary staging in breast cancer patients.

Handheld array-based photoacoustic probe for guiding needle biopsy of sentinel lymph nodes

By modifying a clinical ultrasound array system, we develop a novel handheld photoacoustic probe for image-guided needle biopsy. The integration of optical fiber bundles for pulsed laser light delivery enables photoacoustic image-guided insertion of a needle into rat axillary lymph nodes with accumulated indocyanine green (ICG). Strong photoacoustic contrast of the needle is achieved. After subcutaneous injection of the dye in the left forepaw, sentinel lymph nodes are easily detected, in vivo and in real time, beneath 2-cm-thick chicken breast overlaying the axillary region. ICG uptake in axillary lymph nodes is confirmed with fluorescence imaging both in vivo and ex vivo. These results demonstrate the clinical potential of this handheld photoacoustic system for facile identification and needle biopsy of sentinel lymph nodes for cancer staging and metastasis detection in humans.

Quantitative photoacoustic imaging: correcting for heterogeneous light fluence distributions using diffuse optical tomography

The specificity of molecular and functional photoacoustic (PA) images depends on the accuracy of the photoacoustic absorption spectroscopy. The PA signal is proportional to the product of the optical absorption coefficient and local light fluence; quantitative PA measurements of the optical absorption coefficient therefore require an accurate estimation of optical fluence. Light-modeling aided by diffuse optical tomography (DOT) can be used to map the required fluence and to reduce errors in traditional PA spectroscopic analysis. As a proof-of-concept, we designed a tissue-mimicking phantom to demonstrate how fluence-related artifacts in PA images can lead to misrepresentations of tissue properties. To correct for these inaccuracies, the internal fluence in the tissue phantom was estimated by using DOT to reconstruct spatial distributions of the absorption and reduced scattering coefficients of multiple targets within the phantom. The derived fluence map, which only consisted of low spatial frequency components, was used to correct PA images of the phantom. Once calibrated to a known absorber, this method reduced errors in estimated absorption coefficients from 33% to 6%. These results experimentally demonstrate that combining DOT with PA imaging can significantly reduce fluence-related errors in PA images, while producing quantitatively accurate, high-resolution images of the optical absorption coefficient.

Performance benchmarks of an array-based hand-held photoacoustic probe adapted from a clinical ultrasound system for non-invasive sentinel lymph node imaging.

Clinical translation of photoacoustic (PA) imaging can be facilitated by integration with commercial ultrasound (US) scanners to enable dual-modality imaging. An array-based US scanner was modified for hand-held PA imaging. The performance was benchmarked in terms of signal-to-noise ratio (SNR), axial spatial resolution and sensitivity. PA images of a tube, filled with methylene blue (MB; approx. 30 mM) and placed at various depths in chicken tissue, were acquired. A 5 cm penetration depth was achieved with an 18.6 dB SNR using a laser fluence of 3 mJ cm−2, only one-seventh of the safety limit (20 mJ cm−2). An axial resolution of approximately 400 μm was maintained at all imaging depths. The PA sensitivity to MB placed 2.3 cm deep in chicken tissue was less than 100 μM. Further, after intradermal injection of MB (approx. 30 mM), a rat sentinel lymph node was clearly identified in vivo, beneath a 3.8 cm thick layer of chicken breast. The accumulated concentration of MB in the node was estimated to be approximately 7 mM. The noise-equivalent sensitivities (approx. 2 cm depth) were 17 and 85 μM, ex vivo and in vivo, respectively. These results support the use of this PA system for non-invasive mapping and image-guided needle biopsy of sentinel nodes in breast cancer patients.

High Sensitivity : High-Resolution SPECT-CT/MR Molecular Imaging of Angiogenesis in the Vx2 Model

Objectives:The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality &agr;&ngr;&bgr;3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis. Materials and Methods:In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled &agr;&ngr;&bgr;3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity – high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping. Results:&agr;&ngr;&bgr;3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 ± 0.13, TMR) versus the 11MBq/kg (7.32 ± 0.12) and 44 MBq/kg (6.55 ± 0.07) doses, (P < 0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 ± 0.09) was less (P < 0.05) than the equivalent dosage of &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc &agr;&ngr;&bgr;3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P < 0.05) TMR (5.31 ± 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc &agr;&ngr;&bgr;3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using &agr;&ngr;&bgr;3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer. Conclusion:Dual modality molecular imaging with &agr;&ngr;&bgr;3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Dual-Modality Photoacoustic and Ultrasound Imaging System for Noninvasive Sentinel Lymph Node Detection in Patients with Breast Cancer

The detection of regional lymph node metastases is important in cancer staging as it guides the prognosis of the patient and the strategy for treatment. Sentinel lymph node biopsy (SLNB) is an accurate, less invasive alternative to axillary lymph node dissection. The sentinel lymph node hypothesis states that the pathological status of the axilla can be accurately predicted by determining the status of the first lymph nodes that drain from the primary tumor. Physicians use radio-labeled sulfur colloid and/or methylene blue dye to identify the SLN, which is most likely to contain metastatic cancer cells. However, the surgical procedure causes morbidity and associated expenses. To overcome these limitations, we developed a dual-modality photoacoustic and ultrasonic imaging system to noninvasively detect SLNs based on the accumulation of methylene blue dye. Ultimately, we aim to guide percutaneous needle biopsies and provide a minimally invasive method for axillary staging of breast cancer.

Multi-Scale Molecular Photoacoustic Tomography of Gene Expression

Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ∼1.0 mm and ∼0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 µm and an axial resolution of 15 µm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.

Performance characterization of an integrated ultrasound, photoacoustic, and thermoacoustic imaging system

We developed a novel trimodality system for human breast imaging by integrating photoacoustic (PA) and thermoacoustic (TA) imaging techniques into a modified commercial ultrasound scanner. Because light was delivered with an optical assembly placed within the microwave antenna, no mechanical switching between the microwave and laser sources was needed. Laser and microwave excitation pulses were interleaved to enable PA and TA data acquisition in parallel at a rate of 10 frames per second. A tube (7 mm inner diameter) filled with oxygenated bovine blood or 30 mM methylene blue dye was successfully detected in PA images in chicken breast tissue at depths of 6.6 and 8.4 cm, respectively, for the first time. The SNRs at these depths reached ∼24 and ∼15 dB, respectively, by averaging 200 signal acquisitions. Similarly, a tube (13 mm inner diameter) filled with saline solution (0.9%) at a depth of 4.4 cm in porcine fat tissue was successfully detected in TA images. The PA axial, lateral, and elevational resolutions were 640 μm, 720 μm, and 3.5 mm, respectively, suitable for breast cancer imaging. A PA noise-equivalent sensitivity to methylene blue solution of 260 nM was achieved in chicken tissue at a depth of 3.4 cm.

In vivo three-dimensional photoacoustic imaging based on a clinical matrix array ultrasound probe

We present an integrated photoacoustic and ultrasonic three-dimensional (3-D) volumetric imaging system based on a two-dimensional (2-D) matrix array ultrasound probe. A wavelength-tunable dye laser pumped by a Q-switched Nd:YAG laser serves as the light source and a modified commercial ultrasound imaging system (iU22, Philips Healthcare) with a 2-D array transducer (X7-2, Philips Healthcare) detects both the pulse-echo ultrasound and photoacoustic signals. A multichannel data acquisition system acquires the RF channel data. The imaging system enables rendering of co-registered 3-D ultrasound and photoacoustic images without mechanical scanning. The resolution along the azimuth, elevation, and axial direction are measured to be 0.69, 0.90 and 0.84 mm for photoacoustic imaging. In vivo 3-D photoacoustic mapping of the sentinel lymph node was demonstrated in a rat model using methylene blue dye. These results highlight the clinical potential of 3-D PA imaging for identification of sentinel lymph nodes for cancer staging in humans.