Biomimetic-Membrane-Protected Plasmonic Nanostructures as Dual-Modality Contrast Agents for Correlated Surface-Enhanced Raman Scattering and Photoacoustic Detection of Hidden Tumor Lesions.

Optical imaging and spectroscopic modalities are of considerable current interest for in vivo cancer detection and image-guided surgery, but the turbid or scattering nature of biomedical tissues has severely limited their abilities to detect buried or occluded tumor lesions. Here we report the development of a dual-modality plasmonic nanostructure based on colloidal gold nanostars (AuNSs) for simultaneous surface-enhanced Raman scattering (SERS) and photoacoustic (PA) detection of tumor phantoms embedded (hidden) in ex vivo animal tissues. By using red blood cell membranes as a naturally derived biomimetic coating, we show that this class of dual-modality contrast agents can provide both Raman spectroscopic and PA signals for the detection and differentiation of hidden solid tumors with greatly improved depths of tissue penetration. Compared to previous polymer-coated AuNSs, the biomimetic coatings are also able to minimize protein adsorption and cellular uptake when exposed to human plasma without compromising their SERS or PA signals. We further show that tumor-targeting peptides (such as cyclic RGD) can be noncovalently inserted for targeting the ανß3-integrin receptors expressed on metastatic cancer cells and tracked via both SERS and PA imaging (PAI). Finally, we demonstrate image-guided resections of tumor-mimicking phantoms comprising metastatic tumor cells buried under layers of skin and fat tissues (6 mm in thickness). Specifically, PAI was used to determine the precise tumor location, while SERS spectroscopic signals were used for tumor identification and differentiation. This work opens the possibility of using these biomimetic dual-modality nanoparticles with superior signal and biological stability for intraoperative cancer detection and resection.

Bioinspired, vertically stacked, and perovskite nanocrystal-enhanced CMOS imaging sensors for resolving UV spectral signatures.

Imaging and identifying target signatures and biomedical markers in the ultraviolet (UV) spectrum is broadly important to medical imaging, military target tracking, remote sensing, and industrial automation. However, current silicon-based imaging sensors are fundamentally limited because of the rapid absorption and attenuation of UV light, hindering their ability to resolve UV spectral signatures. Here, we present a bioinspired imaging sensor capable of wavelength-resolved imaging in the UV range. Inspired by the UV-sensitive visual system of the Papilio xuthus butterfly, the sensor monolithically combines vertically stacked photodiodes and perovskite nanocrystals. This imaging design combines two complementary UV detection mechanisms: The nanocrystal layer converts a portion of UV signals into visible fluorescence, detected by the photodiode array, while the remaining UV light is detected by the top photodiode. Our label-free UV fluorescence imaging data from aromatic amino acids and cancer/normal cells enables real-time differentiation of these biomedical materials with 99% confidence.

Fluorescence-guided surgical system using holographic display: from phantom studies to canine patients.

Significance: Holographic display technology is a promising area of research that can lead to significant advancements in cancer surgery. We present the benefits of combining bioinspired multispectral imaging technology with holographic goggles for fluorescence-guided cancer surgery. Through a series of experiments with 43D-printed phantoms, small animal models of cancer, and surgeries on canine patients with head and neck cancer, we showcase the advantages of this holistic approach. Aim: The aim of our study is to demonstrate the feasibility and potential benefits of utilizing holographic display for fluorescence-guided surgery through a series of experiments involving 3D-printed phantoms and canine patients with head and neck cancer. Approach: We explore the integration of a bioinspired camera with a mixed reality headset to project fluorescent images as holograms onto a see-through display, and we demonstrate the potential benefits of this technology through benchtop and in vivo animal studies. Results: Our complete imaging and holographic display system showcased improved delineation of fluorescent targets in phantoms compared with the 2D monitor display approach and easy integration into the veterinarian surgical workflow. Conclusions: Based on our findings, it is evident that our comprehensive approach, which combines a bioinspired multispectral imaging sensor with holographic goggles, holds promise in enhancing the presentation of fluorescent information to surgeons during intraoperative scenarios while minimizing disruptions.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυß3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

Bioinspired color-near infrared endoscopic imaging system for molecular guided cancer surgery.

Significance: Fluorescently guided minimally invasive surgery is improving patient outcomes and disease-free survival, but biomarker variability hinders complete tumor resection with single molecular probes. To overcome this, we developed a bioinspired endoscopic system that images multiple tumor-targeted probes, quantifies volumetric ratios in cancer models, and detects tumors in ex vivo samples. Aim: We present a new rigid endoscopic imaging system (EIS) that can capture color images while simultaneously resolving two near-infrared (NIR) probes. Approach: Our optimized EIS integrates a hexa-chromatic image sensor, a rigid endoscope optimized for NIR-color imaging, and a custom illumination fiber bundle. Results: Our optimized EIS achieves a 60% improvement in NIR spatial resolution when compared to a leading FDA-approved endoscope. Ratio-metric imaging of two tumor-targeted probes is demonstrated in vials and animal models of breast cancer. Clinical data gathered from fluorescently tagged lung cancer samples on the operating room's back table demonstrate a high tumor-to-background ratio and consistency with the vial experiments. Conclusions: We investigate key engineering breakthroughs for the single-chip endoscopic system, which can capture and distinguish numerous tumor-targeting fluorophores. As the molecular imaging field shifts toward a multi-tumor targeted probe methodology, our imaging instrument can aid in assessing these concepts during surgical procedures.

Protease-activated indocyanine green nanoprobes for intraoperative NIR fluorescence imaging of primary tumors.

Tumor-targeted fluorescent probes in the near-infrared spectrum can provide invaluable information about the location and extent of primary and metastatic tumors during intraoperative procedures to ensure no residual tumors are left in the patient's body. Even though the first fluorescence-guided surgery was performed more than 50 years ago, it is still not accepted as a standard of care in part due to the lack of efficient and non-toxic targeted probes approved by regulatory agencies around the world. Herein, we report protease-activated cationic gelatin nanoparticles encapsulating indocyanine green (ICG) for the detection of primary breast tumors in murine models with high tumor-to-background ratios. Upon intravenous administration, these nanoprobes remain optically silent due to the energy resonance transfer among the bound ICG molecules. As the nanoprobes extravasate and are exposed to the acidic tumor microenvironment, their positive surface charges increase, facilitating cellular uptake. The internalized nanoprobes are activated upon proteolytic degradation of gelatin to allow high contrast between the tumor and normal tissue. Since both gelatin and ICG are FDA-approved for intravenous administration, this activatable nanoprobe can lead to quick clinical adoption and improve the treatment of patients undergoing image-guided cancer surgery.

Decoupling channel count from field of view and spatial resolution in single-sensor imaging systems for fluorescence image-guided surgery.

Significance: Near-infrared fluorescence image-guided surgery is often thought of as a spectral imaging problem where the channel count is the critical parameter, but it should also be thought of as a multiscale imaging problem where the field of view and spatial resolution are similarly important. Aim: Conventional imaging systems based on division-of-focal-plane architectures suffer from a strict relationship between the channel count on one hand and the field of view and spatial resolution on the other, but bioinspired imaging systems that combine stacked photodiode image sensors and long-pass/short-pass filter arrays offer a weaker tradeoff. Approach: In this paper, we explore how the relevant changes to the image sensor and associated image processing routines affect image fidelity during image-guided surgeries for tumor removal in an animal model of breast cancer and nodal mapping in women with breast cancer. Results: We demonstrate that a transition from a conventional imaging system to a bioinspired one, along with optimization of the image processing routines, yields improvements in multiple measures of spectral and textural rendition relevant to surgical decision-making. Conclusions: These results call for a critical examination of the devices and algorithms that underpin image-guided surgery to ensure that surgeons receive high-quality guidance and patients receive high-quality outcomes as these technologies enter clinical practice.

Biomimetic Surface-Enhanced Raman Scattering Nanoparticles with Improved Dispersibility, Signal Brightness, and Tumor Targeting Functions.

The development of biocompatible and nontoxic surface-enhanced Raman scattering (SERS) nanoparticles is of considerable current interest because of their attractive biomedical applications such as ultrasensitive in vitro diagnostics, in vivo tumor imaging, and spectroscopy-guided cancer surgery. However, current SERS nanoparticles are prepared and stored in aqueous solution, have limited stability and dispersibility, and are not suitable for lyophilization and storage by freeze-drying or other means. Here, we report a simple but robust method to coat colloidal SERS nanoparticles by naturally derived biomimetic red blood cell membranes (RBCM), leading to a dramatic improvement in stability and dispersibility under freeze-thawing, lyophilization, heating, and physiological conditions. The results demonstrate that the lyophilized SERS nanoparticles in the solid form can be readily dissolved and dispersed in physiological buffer solutions. A surprising finding is that the RBCM-coated SERS particles are considerably brighter (by as much as 5-fold) than PEGylated SERS particles under similar experimental conditions. This additional enhancement is believed to arise from the hydrophobic nature of RBCM's hydrocarbon chains, which is known to reduce electronic dampening and boost electromagnetic field enhancement. A further advantage in using biomimetic membrane coatings is that the bilayer membrane structure allows nonvalent insertion of molecular ligands for tumor targeting. In particular, we show that cyclic-RGD, a tumor-targeting peptide, can be efficiently inserted into the membrane coatings of SERS nanoparticles for targeting the ανß3 integrin receptors expressed on cancer cells. Thus, biomimetic RBCMs provide major advantages over traditional polyethylene glycols for preparing SERS nanoparticles with improved dispersibility, higher signal intensity, and more efficient biofunctionalization.

Hexachromatic bioinspired camera for image-guided cancer surgery.

Cancer affects one in three people worldwide. Surgery remains the primary curative option for localized cancers, but good prognoses require complete removal of primary tumors and timely recognition of metastases. To expand surgical capabilities and enhance patient outcomes, we developed a six-channel color/near-infrared image sensor inspired by the mantis shrimp visual system that enabled near-infrared fluorescence image guidance during surgery. The mantis shrimp's unique eye, which maximizes the number of photons contributing to and the amount of information contained in each glimpse of its surroundings, is recapitulated in our single-chip imaging system that integrates arrays of vertically stacked silicon photodetectors and pixelated spectral filters. To provide information about tumor location unavailable from a single instrument, we tuned three color channels to permit an intuitive perspective of the surgical procedure and three near-infrared channels to permit multifunctional imaging of optical probes highlighting cancerous tissue. In nude athymic mice bearing human prostate tumors, our image sensor enabled simultaneous detection of two tumor-targeted fluorophores, distinguishing diseased from healthy tissue in an estimated 92% of cases. It also permitted extraction of near-infrared structured illumination enabling the mapping of the three-dimensional topography of tumors and surgical sites to within 1.2-mm error. In the operating room, during surgical resection in 18 patients with breast cancer, our image sensor further enabled sentinel lymph node mapping using clinically approved near-infrared fluorophores. The flexibility and performance afforded by this simple and compact architecture highlights the benefits of biologically inspired sensors in image-guided surgery.

Bio-inspired imager improves sensitivity in near-infrared fluorescence image-guided surgery.

Image-guided surgery can enhance cancer treatment by decreasing, and ideally eliminating, positive tumor margins and iatrogenic damage to healthy tissue. Current state-of-the-art near-infrared fluorescence imaging systems are bulky and costly, lack sensitivity under surgical illumination, and lack co-registration accuracy between multimodal images. As a result, an overwhelming majority of physicians still rely on their unaided eyes and palpation as the primary sensing modalities for distinguishing cancerous from healthy tissue. Here we introduce an innovative design, comprising an artificial multispectral sensor inspired by the Morpho butterfly's compound eye, which can significantly improve image-guided surgery. By monolithically integrating spectral tapetal filters with photodetectors, we have realized a single-chip multispectral imager with 1000 × higher sensitivity and 7 × better spatial co-registration accuracy compared to clinical imaging systems in current use. Preclinical and clinical data demonstrate that this technology seamlessly integrates into the surgical workflow while providing surgeons with real-time information on the location of cancerous tissue and sentinel lymph nodes. Due to its low manufacturing cost, our bio-inspired sensor will provide resource-limited hospitals with much-needed technology to enable more accurate value-based health care.

Optical characterization of rigid endoscopes and polarization calibration methods.

Polarization imaging can reveal orthogonal information with respect to color about the structural composition of biological tissue, and with the advance of superior polarimeters its use for biomedical applications has proliferated in the last decade. Polarimetry can be used in pre-clinical and clinical settings for the early detection of cancerous tissue. Polarization-based endoscopy with the complementary near-infrared fluorescence imaging modality improves the early diagnosis of flat cancerous lesions in colorectal tumor models. With the development of new polarization sensors the need to use standard laboratory optics to create custom imaging systems increases. These additional optics can behave as polarization filters effectively degrading and modifying the original tissue's polarization signatures leading to erroneous judgments. Here, we present a framework to characterize the spectral and polarization properties of rigid endoscopes for polarization-based endoscopic imaging. We describe and evaluate two calibration schemes based on Mueller calculus to reconstruct the original polarization information. Optical limitations of the endoscopes and minimum polarimeter requirements are discussed that may be of interest to other researchers working with custom polarization-based imaging systems.

Optical See-Through Cancer Vision Goggles Enable Direct Patient Visualization and Real-Time Fluorescence-Guided Oncologic Surgery.

BACKGROUND: The inability to visualize the patient and surgical site directly, limits the use of current near infrared fluorescence-guided surgery systems for real-time sentinel lymph node biopsy and tumor margin assessment. METHODS: We evaluated an optical see-through goggle augmented imaging and navigation system (GAINS) for near-infrared, fluorescence-guided surgery. Tumor-bearing mice injected with a near infrared cancer-targeting agent underwent fluorescence-guided, tumor resection. Female Yorkshire pigs received hind leg intradermal indocyanine green injection and underwent fluorescence-guided, popliteal lymph node resection. Four breast cancer patients received 99mTc-sulfur colloid and indocyanine green retroareolarly before undergoing sentinel lymph node biopsy using radioactive tracking and fluorescence imaging. Three other breast cancer patients received indocyanine green retroareolarly before undergoing standard-of-care partial mastectomy, followed by fluorescence imaging of resected tumor and tumor cavity for margin assessment. RESULTS: Using near-infrared fluorescence from the dyes, the optical see-through GAINS accurately identified all mouse tumors, pig lymphatics, and four pig popliteal lymph nodes with high signal-to-background ratio. In 4 human breast cancer patients, 11 sentinel lymph nodes were identified with a detection sensitivity of 86.67 ± 0.27% for radioactive tracking and 100% for GAINS. Tumor margin status was accurately predicted by GAINS in all three patients, including clear margins in patients 1 and 2 and positive margins in patient 3 as confirmed by paraffin-embedded section histopathology. CONCLUSIONS: The optical see-through GAINS prototype enhances near infrared fluorescence-guided surgery for sentinel lymph node biopsy and tumor margin assessment in breast cancer patients without disrupting the surgical workflow in the operating room.

Compact wearable dual-mode imaging system for real-time fluorescence image-guided surgery.

A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.

Binocular Goggle Augmented Imaging and Navigation System provides real-time fluorescence image guidance for tumor resection and sentinel lymph node mapping.

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence, and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery, and improve the accuracy of cancer staging.

Image overlay solution based on threshold detection for a compact near infrared fluorescence goggle system.

Near infrared (NIR) fluorescence imaging has shown great potential for various clinical procedures, including intraoperative image guidance. However, existing NIR fluorescence imaging systems either have a large footprint or are handheld, which limits their usage in intraoperative applications. We present a compact NIR fluorescence imaging system (NFIS) with an image overlay solution based on threshold detection, which can be easily integrated with a goggle display system for intraoperative guidance. The proposed NFIS achieves compactness, light weight, hands-free operation, high-precision superimposition, and a real-time frame rate. In addition, the miniature and ultra-lightweight light-emitting diode tracking pod is easy to incorporate with NIR fluorescence imaging. Based on experimental evaluation, the proposed NFIS solution has a lower detection limit of 25 nM of indocyanine green at 27 fps and realizes a highly precise image overlay of NIR and visible images of mice in vivo. The overlay error is limited within a 2-mm scale at a 65-cm working distance, which is highly reliable for clinical study and surgical use.

Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer.

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6 ± 1.65) and flat lesions (12.1 ± 1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.42) Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

Real-time fluorescence image-guided oncologic surgery.

Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional-imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high-operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low-cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infrared (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity.

Engineering light-emitting diode surgical light for near-infrared fluorescence image-guided surgical systems.

The near-infrared (NIR) fluorescence signal in the 700 to 900 nm from molecular probes used in fluorescence image-guided surgery (FIGS) is usually weak compared to the NIR component from white light-emitting diode surgical light, which is typically switched off during FIGS to enhance the molecular fluorescence contrast of the image. We propose a simple solution to this critical issue in FIGS by removing NIR light from surgical light with a low cost commercial 3M cool mirror film 330.

Dual-mode optical imaging system for fluorescence image-guided surgery.

In this Letter, we present a novel imaging concept that a single imaging system can image different spectral bands with different aperture sizes. It is achieved by using a filter with different transmitted spectral bands in different annular rings as the aperture stop. This concept will enable more efficient system configurations and practical clinical applications. We have demonstrated this concept with a dual-mode near-infrared fluorescence image guided surgical system.

Bioinspired Polarization Imaging Sensors: From Circuits and Optics to Signal Processing Algorithms and Biomedical Applications: Analysis at the focal plane emulates nature's method in sensors to image and diagnose with polarized light.

In this paper, we present recent work on bioinspired polarization imaging sensors and their applications in biomedicine. In particular, we focus on three different aspects of these sensors. First, we describe the electro-optical challenges in realizing a bioinspired polarization imager, and in particular, we provide a detailed description of a recent low-power complementary metal-oxide-semiconductor (CMOS) polarization imager. Second, we focus on signal processing algorithms tailored for this new class of bioinspired polarization imaging sensors, such as calibration and interpolation. Third, the emergence of these sensors has enabled rapid progress in characterizing polarization signals and environmental parameters in nature, as well as several biomedical areas, such as label-free optical neural recording, dynamic tissue strength analysis, and early diagnosis of flat cancerous lesions in a murine colorectal tumor model. We highlight results obtained from these three areas and discuss future applications for these sensors.