Dual-Function Antibody Conjugate-Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) for head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual-function antibody conjugate (DFAC) for multimodal imaging and photoimmunotherapy (PIT) of EGFR-overexpressing cancer cells. The DFAC reported previously and used in the present study comprises an EGFR-targeted antibody, cetuximab, conjugated to benzoporphyrin derivative (BPD) for fluorescence imaging and PIT and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3) developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response, with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess the presence of tumor on a macroscopic scale, followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intraoperative setting.

Optimizing Axial and Peripheral Substitutions in Si-Centered Naphthalocyanine Dyes for Enhancing Aqueous Solubility and Photoacoustic Signal Intensity.

Photoacoustic imaging using external contrast agents is emerging as a powerful modality for real-time molecular imaging of deep-seated tumors. There are several chromophores, such as indocyanine green and IRDye800, that can potentially be used for photoacoustic imaging; however, their use is limited due to several drawbacks, particularly photostability. There is, therefore, an urgent need to design agents to enhance contrast in photoacoustic imaging. Naphthalocyanine dyes have been demonstrated for their use as photoacoustic contrast agents; however, their low solubility in aqueous solvents and high aggregation propensity limit their application. In this study, we report the synthesis and characterization of silicon-centered naphthalocyanine dyes with high aqueous solubility and near infra-red (NIR) absorption in the range of 850-920 nm which make them ideal candidates for photoacoustic imaging. A series of Silicon-centered naphthalocyanine dyes were developed with varying axial and peripheral substitutions, all in an attempt to enhance their aqueous solubility and improve photophysical properties. We demonstrate that axial incorporation of charged ammonium mesylate group enhances water solubility. Moreover, the incorporation of peripheral 2-methoxyethoxy groups at the α-position modulates the electronic properties by altering the π-electron delocalization and enhancing photoacoustic signal amplitude. In addition, all the dyes were synthesized to incorporate an N-hydroxysuccinimidyl group to enable further bioconjugation. In summary, we report the synthesis of water-soluble silicon-centered naphthalocyanine dyes with a high photoacoustic signal amplitude that can potentially be used as contrast agents for molecular photoacoustic imaging.

Correction: Bulsink et al. Oxygen Saturation Imaging Using LED-Based Photoacoustic System. Sensors 2021, 21, 283.

The authors wish to make the following corrections to this paper [...].

Oxygen Saturation Imaging Using LED-Based Photoacoustic System.

Oxygen saturation imaging has potential in several preclinical and clinical applications. Dual-wavelength LED array-based photoacoustic oxygen saturation imaging can be an affordable solution in this case. For the translation of this technology, there is a need to improve its accuracy and validate it against ground truth methods. We propose a fluence compensated oxygen saturation imaging method, utilizing structural information from the ultrasound image, and prior knowledge of the optical properties of the tissue with a Monte-Carlo based light propagation model for the dual-wavelength LED array configuration. We then validate the proposed method with oximeter measurements in tissue-mimicking phantoms. Further, we demonstrate in vivo imaging on small animal and a human subject. We conclude that the proposed oxygen saturation imaging can be used to image tissue at a depth of 6-8 mm in both preclinical and clinical applications.

Photochemical Tissue Passivation of Arteriovenous Grafts Prevents Long-Term Development of Intimal Hyperplasia in a Swine Model.

BACKGROUND: The autologous vein remains the standard conduit for lower extremity and coronary artery bypass grafting despite a 30%-50% 5-y failure rate, primarily attributable to intimal hyperplasia (IH) that develops in the midterm period (3-24 mo) of graft maturation. Our group discovered that externally strengthening vein grafts by cross-linking the adventitial collagen with photochemical tissue passivation (PTP) mitigates IH in an arteriovenous model at 4 wk. We now investigate whether this effect is retained in the midterm period follow-up. METHODS: Six Hanford miniature pigs received bilateral carotid artery interposition vein grafts. In each animal, the external surface of one graft was treated with PTP before grafting, whereas the opposite side served as the untreated control. The grafts were harvested after 3 mo. Ultrasound evaluation of all vein grafts was performed at the time of grafting and harvest. The grafts were also evaluated histomorphometrically and immunohistologically for markers of IH. RESULTS: All vein grafts were patent at 3 mo except one graft in the PTP-treated group because of early technical failure. The control vein grafts had significantly greater IH than PTP-treated grafts at 3 mo, as evidenced by the intimal area (2.6 ± 1.0 mm2versus 1.4 ± 1.5 mm2, respectively, P = 0.045) and medial area (5.1 ± 1.9 mm2versus 2.7 ± 2.4 mm2, respectively, P = 0.048). The control grafts had an increased presence and proliferation of mural myofibroblasts with greater smooth muscle actin and proliferating cell nuclear antigen staining. CONCLUSIONS: PTP treatment to the external surface of the vein grafts decreases IH at 3 mo after arteriovenous grafting and may prevent future graft failure.

In Vivo Tumor Vascular Imaging with Light Emitting Diode-Based Photoacoustic Imaging System.

Photoacoustic (PA) imaging has shown tremendous promise for imaging tumor vasculature and its function at deeper penetration depths without the use of exogenous contrast agents. Traditional PA imaging systems employ expensive and bulky class IV lasers with low pulse repetition rate, due to which its availability for preclinical cancer research is hampered. In this study, we evaluated the capability of a Light-Emitting Diode (LED)-based PA and ultrasound (US) imaging system for monitoring heterogeneous microvasculature in tumors (up to 10 mm in depth) and quantitatively compared the PA images with gold standard histology images. We used a combination of a 7 MHz linear array US transducer and 850 nm excitation wavelength LED arrays to image blood vessels in a subcutaneous tumor model. After imaging, the tumors were sectioned and stained for endothelial cells to correlate with PA images across similar cross-sections. Analysis of 30 regions of interest in tumors from different mice showed a statistically significant R-value of 0.84 where the areas with high blood vessel density had high PA response while low blood vessel density regions had low PA response. Our results confirm that LED-based PA and US imaging can provide 2D and 3D images of tumor vasculature and the potential it has as a valuable tool for preclinical cancer research.

Optimizing Irradiation Geometry in LED-Based Photoacoustic Imaging with 3D Printed Flexible and Modular Light Delivery System.

Photoacoustic (PA) imaging-a technique combining the ability of optical imaging to probe functional properties of the tissue and deep structural imaging ability of ultrasound-has gained significant popularity in the past two decades for its utility in several biomedical applications. More recently, light-emitting diodes (LED) are being explored as an alternative to bulky and expensive laser systems used in PA imaging for their portability and low-cost. Due to the large beam divergence of LEDs compared to traditional laser beams, it is imperative to quantify the angular dependence of LED-based illumination and optimize its performance for imaging superficial or deep-seated lesions. A custom-built modular 3-D printed hinge system and tissue-mimicking phantoms with various absorption and scattering properties were used in this study to quantify the angular dependence of LED-based illumination. We also experimentally calculated the source divergence of the pulsed-LED arrays to be 58° ± 8°. Our results from point sources (pencil lead phantom) in non-scattering medium obey the cotangential relationship between the angle of irradiation and maximum PA intensity obtained at various imaging depths, as expected. Strong dependence on the angle of illumination at superficial depths (-5°/mm at 10 mm) was observed that becomes weaker at intermediate depths (-2.5°/mm at 20 mm) and negligible at deeper locations (-1.1°/mm at 30 mm). The results from the tissue-mimicking phantom in scattering media indicate that angles between 30-75° could be used for imaging lesions at various depths (12 mm-28 mm) where lower LED illumination angles (closer to being parallel to the imaging plane) are preferable for deep tissue imaging and superficial lesion imaging is possible with higher LED illumination angles (closer to being perpendicular to the imaging plane). Our results can serve as a priori knowledge for the future LED-based PA system designs employed for both preclinical and clinical applications.

Impacting Pancreatic Cancer Therapy in Heterotypic in Vitro Organoids and in Vivo Tumors with Specificity-Tuned, NIR-Activable Photoimmunonanoconjugates: Towards Conquering Desmoplasia?

Despite untiring efforts to develop therapies for pancreatic ductal adenocarcinoma (PDAC), survival statistics remain dismal, necessitating distinct approaches. Photodynamic priming (PDP), which improves drug delivery and combination regimens, as well as tumor photodestruction are key attributes of photodynamic therapy (PDT), making it a distinctive clinical option for PDAC. Localized, high-payload nanomedicine-assisted delivery of photosensitizers (PSs), with molecular specificity and controlled photoactivation, thus becomes critical in order to reduce collateral toxicity during more expansive photodynamic activation procedures with curative intent. As such, targeted photoactivable lipid-based nanomedicines are an ideal candidate but have failed to provide greater than two-fold cancer cell selectivity, if at all, due to their extensive multivariant physical, optical, and chemical complexity. Here, we report (1) a systematic multivariant tuning approach to engineer (Cet, anti-EGFR mAb) photoimmunonanoconjugates (PINs), and (2) stroma-rich heterotypic PDAC in vitro and in vivo models incorporating patient-derived pancreatic cancer-associated fibroblasts (PCAFs) that recapitulate the desmoplasia observed in the clinic. These offer a comprehensive, disease-specific framework for the development of Cet-PINs. Specificity-tuning of the PINs, in terms of PS lipid anchoring, electrostatic modulation, Cet orientation, and Cet surface densities, achieved ∼16-fold binding specificities and rapid penetration of the heterotypic organoids within 1 h, thereby providing a ∼16-fold enhancement in molecular targeted NIR photodestruction. As a demonstration of their inherent amenability for multifunctionality, encapsulation of high payloads of gemcitabine hydrochloride, 5-fluorouracil, and oxaliplatin within the Cet-PINs further improved their antitumor efficacy in the heterotypic organoids. In heterotypic desmoplastic tumors, the Cet-PINs efficiently penetrated up to 470 µm away from blood vessels, and photodynamic activation resulted in substantial tumor necrosis, which was not elicited in T47D tumors (low EGFR) or when using untargeted constructs in both tumor types. Photodynamic activation of the Cet-PINs in the heterotypic desmoplastic tumors resulted in collagen photomodulation, with a 1.5-fold reduction in collagen density, suggesting that PDP may also hold potential for conquering desmoplasia. The in vivo safety profile of photodynamic activation of the Cet-PINs was also substantially improved, as compared to the untargeted constructs. While treatment using the Cet-PINs did not cause any detriment to the mice's health or to healthy proximal tissue, photodynamic activation of untargeted constructs induced severe acute cachexia and weight loss in all treated mice, with substantial peripheral skin necrosis, muscle necrosis, and bowel perforation. This study is the first report demonstrating the true value of molecular targeting for NIR-activable PINs. These constructs integrate high payload delivery, efficient photodestruction, molecular precision, and collagen photomodulation in desmoplastic PDAC tumors in a single treatment using a single construct. Such combined PIN platforms and heterocellular models open up an array of further multiplexed combination therapies to synergistically control desmoplastic tumor progression and extend PDAC patient survival.

Development and evaluation of a low-cost, portable, LED-based device for PDT treatment of early-stage oral cancer in resource-limited settings.

BACKGROUND: Photodynamic therapy (PDT) using δ-aminolevulinic acid (ALA) photosensitization has shown promise in clinical studies for the treatment of early-stage oral malignancies with fewer potential side effects than traditional therapies. Light delivery to oral lesions can also carried out with limited medical infrastructure suggesting the potential for implementation of PDT in global health settings. OBJECTIVES: We sought to develop a cost-effective, battery-powered, fiber-coupled PDT system suitable for intraoral light delivery enabled by smartphone interface and embedded electronics for ease of operation. METHODS: Device performance was assessed in measurements of optical power output, spectral stability, and preclinical assessment of PDT response in ALA-photosensitized squamous carcinoma cell cultures and murine subcutaneous tumor xenografts. RESULTS: The system achieves target optoelectronic performance with a stable battery-powered output of approximately 180 mW from the fiber tip within the desired spectral window for PpIX activation. The device has a compact configuration, user friendly operation and flexible light delivery for the oral cavity. In cell culture, we show that the overall dose-response is consistent with established light sources and complete cell death of ALA photosensitized cells can be achieved in the irradiated zone. In vivo PDT response (reduction in tumor volume) is comparable with a commercial 635 nm laser. CONCLUSIONS: We developed a low-cost, LED-based, fiber-coupled PDT light delivery source that has stable output on battery power and suitable form factor for deployment in rural and/or resource-limited settings. Lasers Surg. Med. 9999:1-7, 2018. © 2018 Wiley Periodicals, Inc.

Photochemical Tissue Passivation Attenuates AV Fistula Intimal Hyperplasia.

OBJECTIVE: We hypothesized that decreasing vein compliance would protect the vein against stretch injury and reduce intimal hyperplasia (IH). BACKGROUND: Although arteriovenous fistulas (AVFs) are the criterion standard for vascular access, their effectiveness is limited by poor patency with 40% to 60% failing due to IH. Venous stretch injury from exposure to arterial pressure induces IH. Photochemical tissue passivation (PTP) crosslinks adventitial collagen, decreasing vein compliance to resemble that of an artery. METHODS: AVFs were created between the femoral artery and epigastric vein in rats (n = 29). PTP was performed on the vein immediately before vessel anastomosis. AVFs were harvested after four weeks. Venous diameter was measured at the initial procedure and harvest. Intimal area was measured for each segment. Ultrasound was performed at harvest to measure AVF flow. RESULTS: Following AVF construction, venous diameter increased by 10% ±â€Š18% for PTP-treated vessels and 78% ±â€Š27% for controls (P ≤ 0.0001). At one month, PTP reduced AVF dilation by 71% compared to control (69% ±â€Š29% vs 241% ±â€Š78%; P ≤ 0.0001). Both juxta-anastomotic intimal area and total intimal area were reduced in PTP-treated vessels compared to control vessels. Specifically, intimal area was 0.024 ±â€Š0.018 and 0.095 ±â€Š0.089 mm for PTP-treated juxta-anastomotic segments of AVF and control, respectively (P < 0.05). Mean total intimal area for PTP-treated and control AVF were 0.080 ±â€Š0.042 and 0.190 ±â€Š0.110 mm, respectively (P < 0.03). AVF flow was 46.9 ±â€Š35.3 and 19.1 ±â€Š10.1 mL/min for PTP-treated and control AVF, respectively (P < 0.109). CONCLUSIONS: These data demonstrate that PTP represents a promising therapy for the prevention of AVF IH, a process that might improve surgical outcomes for patients receiving hemodialysis.

Shining light on the dark side of imaging: excited state absorption enhancement of a bis-styryl BODIPY photoacoustic contrast agent.

A first approach toward understanding the targeted design of molecular photoacoustic contrast agents (MPACs) is presented. Optical and photoacoustic Z-scan spectroscopy was used to identify how nonlinear (excited-state) absorption contributes to enhancing the photoacoustic emission of the curcuminBF2 and bis-styryl (MeOPh)2BODIPY dyes relative to Cy3.

U-Net enhanced real-time LED-based photoacoustic imaging.

Photoacoustic (PA) imaging is hybrid imaging modality with good optical contrast and spatial resolution. Portable, cost-effective, smaller footprint light emitting diodes (LEDs) are rapidly becoming important PA optical sources. However, the key challenge faced by the LED-based systems is the low light fluence that is generally compensated by high frame averaging, consequently reducing acquisition frame-rate. In this study, we present a simple deep learning U-Net framework that enhances the signal-to-noise ratio (SNR) and contrast of PA image obtained by averaging low number of frames. The SNR increased by approximately four-fold for both in-class in vitro phantoms (4.39 ± 2.55) and out-of-class in vivo models (4.27 ± 0.87). We also demonstrate the noise invariancy of the network and discuss the downsides (blurry outcome and failure to reduce the salt & pepper noise). Overall, the developed U-Net framework can provide a real-time image enhancement platform for clinically translatable low-cost and low-energy light source-based PA imaging systems.

Introduction to the Optical Molecular Probes, Imaging and Drug Delivery 2023 feature issue.

A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optical Molecular Probes, Imaging and Drug Delivery conference as part of the Optica Biophotonics Congress in Vancouver, Canada from April 24-27, 2023.

Vascular regional analysis unveils differential responses to anti-angiogenic therapy in pancreatic xenografts through macroscopic photoacoustic imaging.

Pancreatic cancer (PC) is a highly lethal malignancy and the third leading cause of cancer deaths in the U.S. Despite major innovations in imaging technologies, there are limited surrogate radiographic indicators to aid in therapy planning and monitoring. Amongst the various imaging techniques Ultrasound-guided photoacoustic imaging (US-PAI) is a promising modality based on endogenous blood (hemoglobin) and blood oxygen saturation (StO 2 ) contrast to monitor response to anti-angiogenic therapies. Adaptation of US-PAI to the clinical realm requires macroscopic configurations for adequate depth visualization, illuminating the need for surrogate radiographic markers, including the tumoral microvessel density (MVD). In this work, subcutaneous xenografts with PC cell lines AsPC-1 and MIA-PaCa-2 were used to investigate the effects of receptor tyrosine kinase inhibitor (sunitinib) treatment on MVD and StO 2 . Through histological correlation, we have shown that regions of high and low vascular density (HVD and LVD) can be identified through frequency domain filtering of macroscopic PA images which could not be garnered from purely global analysis. We utilized vascular regional analysis (VRA) of treatment-induced StO 2 and total hemoglobin (HbT) changes. VRA as a tool to monitor treatment response allowed us to identify potential timepoints of vascular remodeling, highlighting its ability to provide insights into the TME not only for sunitinib treatment but also other anti-angiogenic therapies.

Multi-spectral photoacoustic imaging combined with acoustic radiation force impulse imaging for applications in tissue engineering.

Tissue engineering is a dynamic field focusing on the creation of advanced scaffolds for tissue and organ regeneration. These scaffolds are customized to their specific applications and are often designed to be complex, large structures to mimic tissues and organs. This study addresses the critical challenge of effectively characterizing these thick, optically opaque scaffolds that traditional imaging methods fail to fully image due to their optical limitations. We introduce a novel multi-modal imaging approach combining ultrasound, photoacoustic, and acoustic radiation force impulse imaging. This combination leverages its acoustic-based detection to overcome the limitations posed by optical imaging techniques. Ultrasound imaging is employed to monitor the scaffold structure, photoacoustic imaging is employed to monitor cell proliferation, and acoustic radiation force impulse imaging is employed to evaluate the homogeneity of scaffold stiffness. We applied this integrated imaging system to analyze melanoma cell growth within silk fibroin protein scaffolds with varying pore sizes and therefore stiffness over different cell incubation periods. Among various materials, silk fibroin was chosen for its unique combination of features including biocompatibility, tunable mechanical properties, and structural porosity which supports extensive cell proliferation. The results provide a detailed mesoscale view of the scaffolds' internal structure, including cell penetration depth and biomechanical properties. Our findings demonstrate that the developed multimodal imaging technique offers comprehensive insights into the physical and biological dynamics of tissue-engineered scaffolds. As the field of tissue engineering continues to advance, the importance of non-ionizing and non-invasive imaging systems becomes increasingly evident, and by facilitating a deeper understanding and better characterization of scaffold architectures, such imaging systems are pivotal in driving the success of future tissue-engineering solutions.

Oxygen-releasing nanodroplets relieve intratumoral hypoxia in head and neck cancer spheroids.

Hypoxia in solid tumors, including head and neck cancer (HNC), contributes to treatment resistance, aggressive phenotypes, and poor clinical outcomes. Perfluorocarbon nanodroplets have emerged as promising oxygen carriers to alleviate tumor hypoxia. However, a thorough characterization of the hypoxia alleviation effects in terms of sustenance of oxygenated environments have not been thoroughly studied. In this study, we developed and characterized perfluoropentane nanodroplets (PFP NDs) for co-delivery of oxygen and the photoactivatable drug or photosensitizer benzoporphyrin derivative (BPD) to hypoxic HNC spheroids. The PFP NDs exhibited excellent stability, efficient oxygen loading/release, and biocompatibility. Using 3D multicellular tumor spheroids of FaDu and SCC9 HNC cells, we demonstrated the ability of oxygenated PFP NDs to penetrate the hypoxic core and alleviate hypoxia, as evidenced by reduced fluorescence of a hypoxia-sensing reagent and downregulation of hypoxia-inducible factors HIF-1α and HIF-2α. BPD-loaded PFP NDs successfully delivered the photosensitizer into the spheroid core in a time-dependent manner. These findings highlight the potential of PFP NDs as a co-delivery platform to overcome hypoxia-mediated treatment resistance and improve therapy outcomes in HNC.

Red light-activated depletion of drug-refractory glioblastoma stem cells and chemosensitization of an acquired-resistant mesenchymal phenotype.

Glioblastoma stem cells (GSCs) are potent tumor initiators resistant to radiochemotherapy, and this subpopulation is hypothesized to re-populate the tumor milieu due to selection following conventional therapies. Here, we show that 5-aminolevulinic acid (ALA) treatment-a pro-fluorophore used for fluorescence-guided cancer surgery-leads to elevated levels of fluorophore conversion in patient-derived GSC cultures, and subsequent red light-activation induces apoptosis in both intrinsically temozolomide chemotherapy-sensitive and -resistant GSC phenotypes. Red light irradiation of ALA-treated cultures also exhibits the ability to target mesenchymal GSCs (Mes-GSCs) with induced temozolomide resistance. Furthermore, sub-lethal light doses restore Mes-GSC sensitivity to temozolomide, abrogating GSC-acquired chemoresistance. These results suggest that ALA is not only useful for fluorescence-guided glioblastoma tumor resection, but that it also facilitates a GSC drug-resistance agnostic, red light-activated modality to mop up the surgical margins and prime subsequent chemotherapy.

Visual inertial odometry enabled 3D ultrasound and photoacoustic imaging.

There is an increasing need for 3D ultrasound and photoacoustic (USPA) imaging technology for real-time monitoring of dynamic changes in vasculature or molecular markers in various malignancies. Current 3D USPA systems utilize expensive 3D transducer arrays, mechanical arms or limited-range linear stages to reconstruct the 3D volume of the object being imaged. In this study, we developed, characterized, and demonstrated an economical, portable, and clinically translatable handheld device for 3D USPA imaging. An off-the-shelf, low-cost visual odometry system (the Intel RealSense T265 camera equipped with simultaneous localization and mapping technology) to track free hand movements during imaging was attached to the USPA transducer. Specifically, we integrated the T265 camera into a commercially available USPA imaging probe to acquire 3D images and compared it to the reconstructed 3D volume acquired using a linear stage (ground truth). We were able to reliably detect 500 µm step sizes with 90.46% accuracy. Various users evaluated the potential of handheld scanning, and the volume calculated from the motion-compensated image was not significantly different from the ground truth. Overall, our results, for the first time, established the use of an off-the-shelf and low-cost visual odometry system for freehand 3D USPA imaging that can be seamlessly integrated into several photoacoustic imaging systems for various clinical applications.

Ultrasound-guided Photoacoustic image Annotation Toolkit in MATLAB (PHANTOM) for preclinical applications.

Depth-dependent fluence-compensation in photoacoustic (PA) imaging is paramount for accurate quantification of chromophores from deep tissues. Here we present a user-friendly toolkit named PHANTOM (PHotoacoustic ANnotation TOolkit for MATLAB) that includes a graphical interface and assists in the segmentation of ultrasound-guided PA images. We modelled the light source configuration with Monte Carlo eXtreme and utilized 3D segmented tissues from ultrasound to generate fluence maps to depth compensate PA images. The methodology was used to analyze PA images of phantoms with varying blood oxygenation and results were validated with oxygen electrode measurements. Two preclinical models, a subcutaneous tumor and a calcified placenta, were imaged and fluence-compensated using the PHANTOM toolkit and the results were verified with immunohistochemistry. The PHANTOM toolkit provides scripts and auxiliary functions to enable biomedical researchers not specialized in optical imaging to apply fluence correction to PA images, enhancing accessibility of quantitative PAI for researchers in various fields.

A Dual Function Antibody Conjugate Enabled Photoimmunotherapy Complements Fluorescence and Photoacoustic Imaging of Head and Neck Cancer Spheroids.

Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) of head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual function antibody conjugate (DFAC) for multi-modal imaging and photoimmunotherapy (PIT) of EGFR over-expressing cancer cells. The DFAC reported previously and used in the present study, comprises of an EGFR targeted antibody - Cetuximab conjugated to Benzoporphyrin derivative (BPD) for fluorescence imaging and PIT, and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3), developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess tumor presence on a macroscopic scale followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intra-operative setting.