KEYWORDS: Acquisition tracking and pointing, Imaging systems, Field programmable gate arrays, Ovary, Tumors, Ultrasonography, Tissue optics, Data acquisition, Photoacoustic spectroscopy, Digital signal processing
Coregistered ultrasound (US) and photoacoustic imaging are emerging techniques for mapping the echogenic anatomical structure of tissue and its corresponding optical absorption. We report a 128-channel imaging system with real-time coregistration of the two modalities, which provides up to 15 coregistered frames per second limited by the laser pulse repetition rate. In addition, the system integrates a compact transvaginal imaging probe with a custom-designed fiber optic assembly for in vivo detection and characterization of human ovarian tissue. We present the coregistered US and photoacoustic imaging system structure, the optimal design of the PC interfacing software, and the reconfigurable field programmable gate array operation and optimization. Phantom experiments of system lateral resolution and axial sensitivity evaluation, examples of the real-time scanning of a tumor-bearing mouse, and ex vivo human ovaries studies are demonstrated.
To develop an indocyanine green (ICG) tracer with slower clearance kinetics, we explored ICG-encapsulating liposomes (Lip) in three different formulations: untargeted (Lip/ICG), targeted to vascular endothelial growth factor (VEGF) receptors (scVEGF-Lip/ICG) by the receptor-binding moiety single-chain VEGF (scVEGF), or decorated with inactivated scVEGF (inactive-Lip/ICG) that does not bind to VEGF receptors. Experiments were conducted with tumor-bearing mice that were placed in a scattering medium with tumors located at imaging depths of either 1.5 or 2.0 cm. Near-infrared fluorescence diffuse optical tomography that provides depth-resolved spatial distributions of fluorescence in tumor was used for the detection of postinjection fluorescent signals. All liposome-based tracers, as well as free ICG, were injected intravenously into mice in the amounts corresponding to 5 nmol of ICG/mouse, and the kinetics of increase and decrease of fluorescent signals in tumors were monitored. A signal from free ICG reached maximum at 15-min postinjection and then rapidly declined with t 1/2 of ∼20 min . The signals from untargeted Lip/ICG and inactive-Lip/ICG also reached maximum at 15-min postinjection, however, declined somewhat slower than free ICG with t 1/2 of ∼30 min . By contrast, a signal from targeted scVEGF-Lip/ICG grew slower than that of all other tracers, reaching maximum at 30-min postinjection and declined much slower than that of other tracers with t 1/2 of ∼90 min , providing a more extended observation window. Higher scVEGF-Lip/ICG tumor accumulation was further confirmed by the analysis of fluorescence on cryosections of tumors that were harvested from animals at 400 min after injection with different tracers.
To overcome the intensive light scattering in biological tissue, diffuse optical tomography (DOT) in the near-infrared range for breast lesion detection is usually combined with other imaging modalities, such as ultrasound, x-ray, and magnetic resonance imaging, to provide guidance. However, these guiding imaging modalities may depend on different contrast mechanisms compared to the optical contrast in the DOT. As a result, they cannot provide reliable guidance for DOT because some lesions may not be detectable by a nonoptical modality but may have a high optical contrast. An imaging modality that relies on optical contrast to provide guidance is desirable for DOT. We present a system that combines a frequency-domain DOT and real-time photoacoustic tomography (PAT) systems to detect and characterize deeply seated targets embedded in a turbid medium. To further improve the contrast, the exogenous contrast agent, indocyanine green (ICG), is used. Our experimental results show that the combined system can detect a tumor-mimicking phantom, which is immersed in intralipid solution with the concentrations ranging from 100 to 10 μM and with the dimensions of 0.8 cm×0.8 cm×0.6 cm , up to 2.5 cm in depth. Mice experiments also confirmed that the combined system can detect tumors and monitor the ICG uptake and washout in the tumor region. This method can potentially improve the accuracy to detect small breast lesions as well as lesions that are sensitive to background tissue changes, such as the lesions located just above the chest wall.
A photoacoustic contrast agent that is based on bis-carboxylic acid derivative of indocyanine green (ICG) covalently conjugated to single-wall carbon nanotubes (ICG/SWCNT) is presented. Covalently attaching ICG to the functionalized SWCNT provides a more robust system that delivers much more ICG to the tumor site. The detection sensitivity of the new contrast agent in a mouse tumor model is demonstrated in vivo by our custom-built photoacoustic imaging system. The summation of the photoacoustic tomography (PAT) beam envelope, referred to as the “PAT summation,” is used to demonstrate the postinjection light absorption of tumor areas in ICG- and ICG/SWCNT-injected mice. It is shown that ICG is able to provide 33% enhancement at approximately 20 min peak response time with reference to the preinjection PAT level, while ICG/SWCNT provides 128% enhancement at 80 min and even higher enhancement of 196% at the end point of experiments (120 min on average). Additionally, the ICG/SWCNT enhancement was mainly observed at the tumor periphery, which was confirmed by fluorescence images of the tumor samples. This feature is highly valuable in guiding surgeons to assess tumor boundaries and dimensions in vivo and to achieve clean tumor margins to improve surgical resection of tumors.
Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs, and fluorescence optical tomography has tremendous potential to provide clinically useful, functional information by identifying tumor hypoxia. The synthesis of a 2-nitroimidazole-indocyanine green conjugate using a piperazine linker (piperazine-2-nitroimidazole-ICG) capable of robust fluorescent imaging of tumor hypoxia is described. In vivo mouse tumor imaging studies were completed and demonstrate an improved imaging capability of the new dye relative to an earlier version of the dye that was synthesized with an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG). Mouse tumors located at imaging depths of 1.5 and 2.0 cm in a turbid medium were imaged at various time points after intravenous injection of the dyes. On average, the reconstructed maximum fluorescence concentration of the tumors injected with piperazine-2-nitroimidazole-ICG was twofold higher than that injected with ethanolamine-2-nitroimidazole-ICG within 3 h postinjection period and 1.6 to 1.7 times higher beyond 3 h postinjection. The untargeted bis-carboxylic acid ICG completely washed out after 3 h postinjection. Thus, the optimal window to assess tumor hypoxia is beyond 3 h postinjection. These findings were supported with fluorescence images of histological sections of tumor samples and an immunohistochemistry technique for identifying tumor hypoxia.
Indocyanine Green encapsulating liposomes (Lip/ICG) and scVEGF-Lip/ICG liposomes, decorated with site-specifically lipidated engineered single-chain vascular endothelial growth factor (scVEGF) for targeting VEGF receptors were tested as potential tracers for fluorescent tomography. Two groups of experiments were conducted with tumor-bearing mice (n=4 to 6 per group) with tumors placed in a scattering medium at the imaging depths of 1.5 and 2.0 cm. Lip/ICG and scVEGF-Lip/ICG were injected intravenously in the amounts corresponding to 5 nmol of ICG/mouse. We detected kinetics of increase and decline in fluorescent signals in tumors for both imaging depths and for both targeted and untargeted Lip/ICG. Maximum fluorescent signals were approximately 2-fold higher at 1.5 cm vs. 2.0 cm imaging. A signal from untargeted Lip/ICG reached maximum at 15 min post-injection and then rapidly declined with t1/2 ~15 min. In contrast, a signal from targeted scVEGF-Lip/ICG reached maximum at 30 min post-injection and then slow declined with t1/2 ~60-90 min. Preferential retention of scVEGF-Lip(ICG) vs. Lip(ICG) was confirmed by the analysis of fluorescence in cryosections of corresponding tumors, harvested at 400 min post-injection. Our results suggest that targeted scVEGF-Lip/ICG can provide for significantly better post-injection time window for detection of relatively deeply seated tumors.
In this paper, we have synthesized a second generation tumor hypoxia targeted 2-nitroimidazole-ICG conjugate using piperazine linker (2-nitro-ICG-p) and validated its performance in in vivo tumor targeting. The results have shown that tumor hypoxia can be targeted with twice higher signal strength beyond three hours post-injection while the un-targeted ICG has completely washed out. The improvement of the second generation 2-nitro-ICG-p dyes is 1.2-1.3 times over the first generation 2-nitro-ICG dyes using ethanol linker beyond 3 hours post-injection which is the optimal time-window for evaluating tumor hypoxia.
In this study, we present a novel photoacoustic contrast agent which is based on bis-carboxylic acid
derivative of Indocyanine green (ICG) covalently conjugated to single-wall carbon nanotubes
(ICG/SWCNT). Covalently attaching ICG to the functionalized SWCNT provides a more robust system
that delivers much more ICG to the tumor site. The detection sensitivity of the new contrast agent in
mouse tumor model is demonstrated in vivo by our custom built photoacoustic imaging system. PAT
summation signal is defined to show the long-term light absorption of tumor areas in ICG injected mice and
ICG/SWCNT injected mice. It is shown that ICG is able to provide 33% enhancement at approximately 20
minutes peak response time referred to pre-injection PAT summation level, while ICG/SWCNT provides
128% enhancement at 80 minutes and even higher enhancement of 196% at the end point of experiments
(120 minutes on average). Additionally, the ICG/SWCNT enhancement was mainly observed at the tumor
periphery as confirmed by fluorescence images of the tumor samples. This feature is highly valuable in
guiding surgeons to assess tumor boundaries and dimensions in vivo and improve surgical resection of
tumors for achieving clean tumor margins.
KEYWORDS: Imaging systems, Acquisition tracking and pointing, Ultrasonography, Field programmable gate arrays, Ovary, Tumors, Tissue optics, In vivo imaging, Pulsed laser operation, Ovarian cancer
In this paper, we report an ultrafast co-registered ultrasound and photoacoustic imaging system based on FPGA parallel
processing. The system features 128-channel parallel acquisition and digitization, along with FPGA-based reconfigurable
processing for real-time co-registered imaging of up to 15 frames per second that is only limited by the laser pulse
repetition frequency of 15 Hz. We demonstrated the imaging capability of the system by live imaging of a mouse tumor
model in vivo, and imaging of human ovaries ex vivo. A compact transvaginal probe that includes the PAT illumination
using a fiber-optic assembly was used for this purpose. The system has the potential ability to assist a clinician to
perform transvaginal ultrasound scanning and to localize the ovarian mass, while simultaneously mapping the light
absorption of the ultrasound detected mass to reveal its vasculature using the co-registered PAT.
We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.
We present a photoacoustic tomography-guided diffuse optical tomography approach using a hand-held probe for detection and characterization of deeply-seated targets embedded in a turbid medium. Diffuse optical tomography guided by coregistered ultrasound, MRI, and x ray has demonstrated a great clinical potential to overcome lesion location uncertainty and to improve light quantification accuracy. However, due to the different contrast mechanisms, some lesions may not be detectable by a nonoptical modality but yet have high optical contrast. Photoacoustic tomography utilizes a short-pulsed laser beam to diffusively penetrate into tissue. Upon absorption of the light by the target, photoacoustic waves are generated and used to reconstruct, at ultrasound resolution, the optical absorption distribution that reveals optical contrast. However, the robustness of optical property quantification of targets by photoacoustic tomography is complicated because of the wide range of ultrasound transducer sensitivity, the orientation and shape of the targets relative to the ultrasound array, and the uniformity of the laser beam. We show in this paper that the relative optical absorption map provided by photoacoustic tomography can potentially guide the diffuse optical tomography to accurately reconstruct target absorption maps.
A handheld photoacoustic tomography-guided diffuse optical tomography system for imaging deeply-seated targets in
scattering media is presented. This hybrid imager consists of a probe with an ultrasound transducer in the center and
straddled by two optical fibers for taking photoacoustic images. The diffuse optical tomography component comprises of
9 light-source fibers for delivering light to the imaged tissue, and 14 detector fibers for collecting the light. Single- and
two-phantom targets of high and low optical contrasts were immersed in a scattering intralipid solution to depths of up to
3cm and imaged. The reconstructed absorption coefficients of the targets with guidance from photoacoustic tomography
were compared to those obtained with a-priori depth-only information, and no a-priori information. The reconstructed
absorption maps yielded as much as 2.6-fold improvement in the quantification accuracy compared to the cases with no
guidance from photoacoustic tomography.
KEYWORDS: Tumors, Carbon nanotubes, Photoacoustic imaging, Photoacoustic spectroscopy, Absorption, Hypoxia, Near infrared, Single walled carbon nanotubes, In vivo imaging, Digital signal processing
Development of new and efficient contrast agents is of fundamental importance to improve detection sensitivity of
smaller lesions. Within the family of nanomaterials, carbon nanotubes (CNT) not only have emerged as a new
alternative and efficient transporter and translocater of therapeutic molecules but also as a photoacoustic molecular
imaging agent owing to its strong optical absorption in the near-infrared region. Drugs, Antibodies and nucleic acids
could functionalize the CNT and prepare an appropriate system for delivering the cargos to cells and organs. In this
work, we present a novel photoacoustic contrast agent which is based on a unique hypoxic marker in the near infrared
region, 2-nitroimidazole -bis carboxylic acid derivative of Indocyanine Green conjugated to single walled carbon
nanotube (SWCNT-2nitroimidazole-ICG). The 2-nitroimidazole-ICG has an absorption peak at 755 nm and an extinction
coefficient of 20,5222 M-1cm-1. The conjugation of this marker with SWCNT shows more than 25 times enhancement of
optical absorption of carbon nanotubes in the near infrared region. This new conjugate has been optically evaluated
and shows promising results for high contrast photoacoustic imaging of deeply located tumors. The conjugate
specifically targets tumor hypoxia, an important indicator of tumor metabolism and tumor therapeutic response. The
detection sensitivity of the new contrast agent has been evaluated in-vitro cell lines and with in-vivo tumors in mice.
We present tumor hypoxia mapping by diffuse optical fluorescence tomography. A novel 2-nitroimidazole bis-carboxylic
acid indocyanine dye conjugate has been developed for tumor-targeted hypoxia fluorescence imaging. The hypoxia
probe has been evaluated in-vitro using 4T1 tumor cell lines and in-vivo tumor targeting in mice. In-vivo tumor targeting
in six mice demonstrated that a measured half-life of 2-nitroimidazole-indocyanine dye wash out in the tumor was
significantly longer (112±32.37 minutes) than that of bis-carboxylic acid indocyanine dye (69.75±14.01 minutes). The
bis-carboxylic acid indocyanine dye was completely washed out from the tumor site within 3-5 hours post-injection, but
2-nitroimidazole-ICG remained for up to 21 hours in the tumor site. Near infrared fluorescence images of mice tumors
showed a 2.6-fold contrast of dye uptake with hypoxic conjugate injection (7.46±1.68 μM) compared to that with
indocyanine dye injection (2.9±0.60 μM). The in-vitro cell studies were performed to assess fluorescence labeling
comparing hypoxia to normoxia conditions. A fluorescence emission ratio of 2.5-fold was found between the cells
treated with the 2-nitroimidazole-indocyanine dye and incubated under hypoxia compared to the cells in normoxia
condition. Hypoxia specificity was also confirmed when compared to cells treated with unconjugated indocyanine dye
alone. Fluorescence images acquired using a Li-COR scanner from harvested tumor samples support the in vivo
monitoring and imaging results.
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