Early Detection of Myeloid-Derived Suppressor Cells in the Lung Pre-Metastatic Niche by Shortwave Infrared Nanoprobes.

Metastatic breast cancer remains a significant source of mortality amongst breast cancer patients and is generally considered incurable in part due to the difficulty in detection of early micro-metastases. The pre-metastatic niche (PMN) is a tissue microenvironment that has undergone changes to support the colonization and growth of circulating tumor cells, a key component of which is the myeloid-derived suppressor cell (MDSC). Therefore, the MDSC has been identified as a potential biomarker for PMN formation, the detection of which would enable clinicians to proactively treat metastases. However, there is currently no technology capable of the in situ detection of MDSCs available in the clinic. Here, we propose the use of shortwave infrared-emitting nanoprobes for the tracking of MDSCs and identification of the PMN. Our rare-earth albumin nanocomposites (ReANCs) are engineered to bind the Gr-1 surface marker of murine MDSCs. When delivered intravenously in murine models of breast cancer with high rates of metastasis, the targeted ReANCs demonstrated an increase in localization to the lungs in comparison to control ReANCs. However, no difference was seen in the model with slower rates of metastasis. This highlights the potential utility of MDSC-targeted nanoprobes to assess PMN development and prognosticate disease progression.

Computational imaging with spectral coding increases the spatial resolution of fiber optic bundles.

Fiber optic bundles are used in narrow-diameter medical and industrial instruments for acquiring images from confined locations. Images transmitted through these bundles contain only one pixel of information per fiber core and fail to capture information from the cladding region between cores. Both factors limit the spatial resolution attainable with fiber bundles. We show here that computational imaging (CI) can be combined with spectral coding to overcome these two fundamental limitations and improve spatial resolution in fiber bundle imaging. By acquiring multiple images of a scene with a high-resolution mask pattern imposed, up to 17 pixels of information can be recovered from each fiber core. A dispersive element at the distal end of the bundle imparts a wavelength-dependent lateral shift on light from the object. This enables light that would otherwise be lost at the inter-fiber cladding to be transmitted through adjacent fiber cores. We experimentally demonstrate this approach using synthetic and real objects. Using CI with spectral coding, object features 5× smaller than individual fiber cores were resolved, whereas conventional imaging could only resolve features at least 1.5× larger than each core. In summary, CI combined with spectral coding provides an approach for overcoming the two fundamental limitations of fiber optic bundle imaging.

Short-Wave Infrared Emitting Nanocomposites for Fluorescence-Guided Surgery.

Fluorescence-guided surgery (FGS) is an emerging technique for tissue visualization during surgical procedures. Structures of interest are labeled with exogenous probes whose fluorescent emissions are acquired and viewed in real-time with optical imaging systems. This study investigated rare-earth-doped albumin-encapsulated nanocomposites (REANCs) as short-wave infrared emitting contrast agents for FGS. Experiments were conducted using an animal model of 4T1 breast cancer. The signal-to-background ratio (SBR) obtained with REANCs was compared to values obtained using indocyanine green (ICG), a near-infrared dye used in clinical practice. Prior to resection, the SBR for tumors following intratumoral administration of REANCs was significantly higher than for tumors injected with ICG. Following FGS, evaluation of fluorescence intensity levels in excised tumors and at the surgical bed demonstrated higher contrast between tissues at these sites with REANC contrast than ICG. REANCs also demonstrated excellent photostability over 2 hours of continuous illumination, as well as the ability to perform FGS under ambient lighting, establishing these nanocomposites as a promising contrast agent for FGS applications.

Shortwave infrared emitting multicolored nanoprobes for biomarker-specific cancer imaging in vivo.

BACKGROUND: The ability to detect tumor-specific biomarkers in real-time using optical imaging plays a critical role in preclinical studies aimed at evaluating drug safety and treatment response. In this study, we engineered an imaging platform capable of targeting different tumor biomarkers using a multi-colored library of nanoprobes. These probes contain rare-earth elements that emit light in the short-wave infrared (SWIR) wavelength region (900-1700 nm), which exhibits reduced absorption and scattering compared to visible and NIR, and are rendered biocompatible by encapsulation in human serum albumin. The spectrally distinct emissions of the holmium (Ho), erbium (Er), and thulium (Tm) cations that constitute the cores of these nanoprobes make them attractive candidates for optical molecular imaging of multiple disease biomarkers. METHODS: SWIR-emitting rare-earth-doped albumin nanocomposites (ReANCs) were synthesized using controlled coacervation, with visible light-emitting fluorophores additionally incorporated during the crosslinking phase for validation purposes. Specifically, HoANCs, ErANCs, and TmANCs were co-labeled with rhodamine-B, FITC, and Alexa Fluor 647 dyes respectively. These Rh-HoANCs, FITC-ErANCs, and 647-TmANCs were further conjugated with the targeting ligands daidzein, AMD3100, and folic acid respectively. Binding specificities of each nanoprobe to distinct cellular subsets were established by in vitro uptake studies. Quantitative whole-body SWIR imaging of subcutaneous tumor bearing mice was used to validate the in vivo targeting ability of these nanoprobes. RESULTS: Each of the three ligand-functionalized nanoprobes showed significantly higher uptake in the targeted cell line compared to untargeted probes. Increased accumulation of tumor-specific nanoprobes was also measured relative to untargeted probes in subcutaneous tumor models of breast (4175 and MCF-7) and ovarian cancer (SKOV3). Preferential accumulation of tumor-specific nanoprobes was also observed in tumors overexpressing targeted biomarkers in mice bearing molecularly-distinct bilateral subcutaneous tumors, as evidenced by significantly higher signal intensities on SWIR imaging. CONCLUSIONS: The results from this study show that tumors can be detected in vivo using a set of targeted multispectral SWIR-emitting nanoprobes. Significantly, these nanoprobes enabled imaging of biomarkers in mice bearing bilateral tumors with distinct molecular phenotypes. The findings from this study provide a foundation for optical molecular imaging of heterogeneous tumors and for studying the response of these complex lesions to targeted therapy.

Shortwave Infrared-Emitting Theranostics for Breast Cancer Therapy Response Monitoring.

Therapeutic drug monitoring (TDM) in cancer, while imperative, has been challenging due to inter-patient variability in drug pharmacokinetics. Additionally, most pharmacokinetic monitoring is done by assessments of the drugs in plasma, which is not an accurate gauge for drug concentrations in target tumor tissue. There exists a critical need for therapy monitoring tools that can provide real-time feedback on drug efficacy at target site to enable alteration in treatment regimens early during cancer therapy. Here, we report on theranostic optical imaging probes based on shortwave infrared (SWIR)-emitting rare earth-doped nanoparticles encapsulated with human serum albumin (abbreviated as ReANCs) that have demonstrated superior surveillance capability for detecting micro-lesions at depths of 1 cm in a mouse model of breast cancer metastasis. Most notably, ReANCs previously deployed for detection of multi-organ metastases resolved bone lesions earlier than contrast-enhanced magnetic resonance imaging (MRI). We engineered tumor-targeted ReANCs carrying a therapeutic payload as a potential theranostic for evaluating drug efficacy at the tumor site. In vitro results demonstrated efficacy of ReANCs carrying doxorubicin (Dox), providing sustained release of Dox while maintaining cytotoxic effects comparable to free Dox. Significantly, in a murine model of breast cancer lung metastasis, we demonstrated the ability for therapy monitoring based on measurements of SWIR fluorescence from tumor-targeted ReANCs. These findings correlated with a reduction in lung metastatic burden as quantified via MRI-based volumetric analysis over the course of four weeks. Future studies will address the potential of this novel class of theranostics as a preclinical pharmacological screening tool.

Shortwave-infrared meso-patterned imaging enables label-free mapping of tissue water and lipid content.

Water and lipids are key participants in many biological processes, but there are few non-invasive methods that provide quantification of these components in vivo, and none that can isolate and quantify lipids in the blood. Here we develop a new imaging modality termed shortwave infrared meso-patterned imaging (SWIR-MPI) to provide label-free, non-contact, spatial mapping of water and lipid concentrations in tissue. The method utilizes patterned hyperspectral illumination to target chromophore absorption bands in the 900-1,300 nm wavelength range. We use SWIR-MPI to monitor clinically important physiological processes including edema, inflammation, and tumor lipid heterogeneity in preclinical models. We also show that SWIR-MPI can spatially map blood-lipids in humans, representing an example of non-invasive and contact-free measurements of in vivo blood lipids. Together, these results highlight the potential of SWIR-MPI to enable new capabilities in fundamental studies and clinical monitoring of major conditions including obesity, cancer, and cardiovascular disease.

An Experimental Review of Optical Coherence Tomography Systems for Noninvasive Assessment of Hard Dental Tissues.

Optical coherence tomography (OCT) is a noninvasive, high-resolution, cross-sectional imaging technique. To date, OCT has been demonstrated in several areas of dentistry, primarily using wavelengths around 1,300 nm, low numerical aperture (NA) imaging lenses, and detectors insensitive to the polarization of light. The objective of this study is to compare the performance of three commercially available OCT systems operating with alternative wavelengths, imaging lenses, and detectors for OCT imaging of dental enamel. Spectral-domain (SD) OCT systems with (i) 840 nm (Lumedica, OQ LabScope 1.0), (ii) 1,300 nm (Thorlabs, Tel320) center wavelengths, and (iii) a swept-source (SS) OCT system (Thorlabs OCS1300SS) centered at 1,325 nm with optional polarization-sensitive detection were used. Low NA (0.04) and high NA (0.15) imaging lenses were used with system (iii). Healthy in vivo and in vitrohuman enamel and eroded in vitro bovine enamel specimens were imaged. The Tel320 system achieved greater imaging depth than the OQ LabScope 1.0, on average imaging 2.6 times deeper into the tooth (n = 10). The low NA lens provided a larger field of view and depth of focus, while the high NA lens provided higher lateral resolution and greater contrast. Polarization-sensitive imaging eliminated birefringent banding artifacts that can appear in conventional OCT scans. In summary, this study illustrates the performance of three commercially available OCT systems, objective lenses, and imaging modes and how these can affect imaging depth, resolution, field of view, and contrast in enamel. Users investigating OCT for dental applications should consider these factors when selecting an OCT system for clinical or basic science studies.

FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.

We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (EFRETapp) of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (R2 = 0.98). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.

Computational endoscopy-a framework for improving spatial resolution in fiber bundle imaging.

This Letter presents a framework for computational imaging (CI) in fiber-bundle-based endoscopy systems. Multiple observations are acquired of objects spatially modulated with different random binary masks. Sparse-recovery algorithms then reconstruct images with more resolved pixels than individual fibers in the bundle. Object details lying within the diameter of single fibers are resolved, allowing images with 41,663 resolvable points to be generated through a bundle with 2,420 fibers. Computational fiber bundle imaging of micro- and macro-scale objects is demonstrated using fluorescent standards and biological tissues, including in vivo imaging of a human fingertip. In each case, CI recovers details that conventional endoscopy does not provide.

Multiscale optical imaging of rare-earth-doped nanocomposites in a small animal model.

Rare-earth-doped nanocomposites have appealing optical properties for use as biomedical contrast agents, but few systems exist for imaging these materials. We describe the design and characterization of (i) a preclinical system for whole animal in vivo imaging and (ii) an integrated optical coherence tomography/confocal microscopy system for high-resolution imaging of ex vivo tissues. We demonstrate these systems by administering erbium-doped nanocomposites to a murine model of metastatic breast cancer. Short-wave infrared emissions were detected in vivo and in whole organ imaging ex vivo. Visible upconversion emissions and tissue autofluorescence were imaged in biopsy specimens, alongside optical coherence tomography imaging of tissue microstructure. We anticipate that this work will provide guidance for researchers seeking to image these nanomaterials across a wide range of biological models.

Optical Projection Tomography with a Tissue Clearing Agent for Developmental and Reproductive Toxicology Studies.

BACKGROUND: Developmental and reproductive toxicology (DART) testing represents an expensive and time-consuming stage in determining the toxicological profile of new chemical entities. Within DART studies, morphological evaluation of fetal skeletons for developmental abnormalities typically requires 7 to 14 days. Current processing techniques involve digestion of soft tissue using a strong base (KOH), followed by qualitative assessment of the remaining skeletal tissue by a fetal morphologist. Micro-computed tomography (micro-CT) has been proposed as a nondestructive image-based alternative for quantitative assessment of skeletal morphology. Such methods eliminate the need for extensive tissue processing and can be paired with quantitative analysis algorithms. However, due to the significant capital and operational expenses required for micro-CT imaging, this approach has yet to gain widespread traction and regulatory acceptance. METHODS: A novel tissue clearing agent was used in 1-week-old rats to render soft tissue optically transparent. Alizarin red was used to stain the skeleton. High dynamic range optical trans-illumination images were then acquired with an optical-CT imaging system and rendered as three-dimensional images of skeletal structure. RESULTS: High dynamic range-based optical-CT imaging of chemically cleared tissues can rapidly generate high resolution (50-250 µm) reconstructions of whole skeletons. CONCLUSION: In summary, this study demonstrates that the combination of tissue clearing, optical imaging, and novel reconstruction algorithms may present a new paradigm for high-throughput evaluation of tissues in DART testing. Birth Defects Research 110:12-16, 2018. © 2017 Wiley Periodicals, Inc.

Vibrational analysis of implants and tissues: Calibration and mechanical spectroscopy of multi-component materials.

Several new methods have been used to non-destructively evaluate the mechanical properties of materials and tissues including magnetic resonance elastography, ultrasound elastography, optical coherence elastography, and various forms of vibrational analysis. One of the limitations of using these methods is the need to establish a relationship between the modulus measured using each new technique and moduli measured using well-established techniques such as constant rate-of-strain and incremental stress-strain curves. In addition, there are no available methods for analyzing the mechanical properties of the individual components of multi-component materials. In this article, we present data showing that there is a strong correlation (correlation coefficient >0.9) between the modulus measured using classical uniaxial tensile incremental stress-strain tests and those made using a combination of optical coherence tomography and vibrational analysis. Beyond this, we demonstrate that the moduli of the major structural components of pig skin can be measured using this technique. These results suggest that optical coherence tomography in concert with vibrational analysis can be used to measure the moduli of biological and implant materials without having to determine Poisson's ratio. In addition, each of the moduli of the major fibrous components of pig skin can be measured concurrently using this technique. These results may be useful in the characterization of synthetic implants and tissue derived materials without requiring removal of one or more components that are to be characterized. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1666-1671, 2017.

A method for nondestructive mechanical testing of tissues and implants.

Numerous tests have been used to elucidate mechanical properties of tissues and implants including tensile, compressive, shear, hydrostatic compression, and three-point bending in one or more axial directions. The development of a nondestructive test that could be applied to tissues and materials in vivo would promote the analysis of tissue pathology as well as the design of implant materials. The purpose of this article is to present the results of preliminary studies demonstrating nondestructive in vitro testing of a tissue model, decellularized human dermis, and a model implant, silicone rubber, using a combination of optical coherence tomography (OCT), and vibrational analysis. The results presented suggest that nondestructive vibrational testing of tissues and materials can be used to determine the modulus of polymeric materials and the results are similar to those found using tensile stress-strain measurements. The advantage of this method is that the modulus can be obtained from vibrational methods without having to approximate the tangent to the stress-strain curve, which is difficult for nonlinear materials that have a rapidly changing slope. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 15-22, 2017.

Surveillance nanotechnology for multi-organ cancer metastases.

The identification and molecular profiling of early metastases remains a major challenge in cancer diagnostics and therapy. Most in vivo imaging methods fail to detect small cancerous lesions, a problem that is compounded by the distinct physical and biological barriers associated with different metastatic niches. Here, we show that intravenously injected rare-earth-doped albumin-encapsulated nanoparticles emitting short-wave infrared light (SWIR) can detect targeted metastatic lesions in vivo, allowing for the longitudinal tracking of multi-organ metastases. In a murine model of basal human breast cancer, the nanoprobes enabled whole-body SWIR detection of adrenal gland microlesions and bone lesions that were undetectable via contrast-enhanced magnetic resonance imaging (CE-MRI) as early as, respectively, three weeks and five weeks post-inoculation. Whole-body SWIR imaging of nanoprobes functionalized to differentially target distinct metastatic sites and administered to a biomimetic murine model of human breast cancer resolved multi-organ metastases that showed varied molecular profiles at the lungs, adrenal glands and bones. Real-time surveillance of lesions in multiple organs should facilitate pre-therapy and post-therapy monitoring in preclinical settings.

Computational imaging with a highly parallel image-plane-coded architecture: challenges and solutions.

This paper investigates a highly parallel extension of the single-pixel camera based on a focal plane array. It discusses the practical challenges that arise when implementing such an architecture and demonstrates that system-specific optical effects must be measured and integrated within the system model for accurate image reconstruction. Three different projection lenses were used to evaluate the ability of the system to accommodate varying degrees of optical imperfection. Reconstruction of binary and grayscale objects using system-specific models and Nesterov's proximal gradient method produced images with higher spatial resolution and lower reconstruction error than using either bicubic interpolation or a theoretical system model that assumes ideal optical behavior. The high-quality images produced using relatively few observations suggest that higher throughput imaging may be achieved with such architectures than with conventional single-pixel cameras. The optical design considerations and quantitative performance metrics proposed here may lead to improved image reconstruction for similar highly parallel systems.

Quantitative analysis of high-resolution microendoscopic images for diagnosis of neoplasia in patients with Barrett's esophagus.

BACKGROUND AND AIMS: Previous studies show that microendoscopic images can be interpreted visually to identify the presence of neoplasia in patients with Barrett's esophagus (BE), but this approach is subjective and requires clinical expertise. This study describes an approach for quantitative image analysis of microendoscopic images to identify neoplastic lesions in patients with BE. METHODS: Images were acquired from 230 sites from 58 patients by using a fiberoptic high-resolution microendoscope during standard endoscopic procedures. Images were analyzed by a fully automated image processing algorithm, which automatically selected a region of interest and calculated quantitative image features. Image features were used to develop an algorithm to identify the presence of neoplasia; results were compared with a histopathology diagnosis. RESULTS: A sequential classification algorithm that used image features related to glandular and cellular morphology resulted in a sensitivity of 84% and a specificity of 85%. Applying the algorithm to an independent validation set resulted in a sensitivity of 88% and a specificity of 85%. CONCLUSIONS: This pilot study demonstrates that automated analysis of microendoscopic images can provide an objective, quantitative framework to assist clinicians in evaluating esophageal lesions from patients with BE. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT01384227 and NCT02018367.).

Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents.

Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4 : Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

Low-Cost High-Resolution Microendoscopy for the Detection of Esophageal Squamous Cell Neoplasia: An International Trial.

BACKGROUND & AIMS: Esophageal squamous cell neoplasia has a high mortality rate as a result of late detection. In high-risk regions such as China, screening is performed by Lugol's chromoendoscopy (LCE). LCE has low specificity, resulting in unnecessary tissue biopsy with a subsequent increase in procedure cost and risk. The purpose of this study was to evaluate the accuracy of a novel, low-cost, high-resolution microendoscope (HRME) as an adjunct to LCE. METHODS: In this prospective trial, 147 consecutive high-risk patients were enrolled from 2 US and 2 Chinese tertiary centers. Three expert and 4 novice endoscopists performed white-light endoscopy followed by LCE and HRME. All optical images were compared with the gold standard of histopathology. RESULTS: By using a per-biopsy analysis, the sensitivity of LCE vs LCE + HRME was 96% vs 91% (P = .0832), specificity was 48% vs 88% (P < .001), positive predictive value was 22% vs 45% (P < .0001), negative predictive value was 98% vs 98% (P = .3551), and overall accuracy was 57% vs 90% (P < .001), respectively. By using a per-patient analysis, the sensitivity of LCE vs LCE + HRME was 100% vs 95% (P = .16), specificity was 29% vs 79% (P < .001), positive predictive value was 32% vs 60%, 100% vs 98%, and accuracy was 47% vs 83% (P < .001). With the use of HRME, 136 biopsies (60%; 95% confidence interval, 53%-66%) could have been spared, and 55 patients (48%; 95% confidence interval, 38%-57%) could have been spared any biopsy. CONCLUSIONS: In this trial, HRME improved the accuracy of LCE for esophageal squamous cell neoplasia screening and surveillance. HRME may be a cost-effective optical biopsy adjunct to LCE, potentially reducing unnecessary biopsies and facilitating real-time decision making in globally underserved regions. ClinicalTrials.gov, NCT 01384708.

Quantitative analysis of high-resolution microendoscopic images for diagnosis of esophageal squamous cell carcinoma.

BACKGROUND & AIMS: High-resolution microendoscopy is an optical imaging technique with the potential to improve the accuracy of endoscopic screening for esophageal squamous neoplasia. Although these microscopic images can be interpreted readily by trained personnel, quantitative image analysis software could facilitate the use of this technology in low-resource settings. In this study, we developed and evaluated quantitative image analysis criteria for the evaluation of neoplastic and non-neoplastic squamous esophageal mucosa. METHODS: We performed an image analysis of 177 patients undergoing standard upper endoscopy for screening or surveillance of esophageal squamous neoplasia, using high-resolution microendoscopy, at 2 hospitals in China and at 1 hospital in the United States from May 2010 to October 2012. Biopsy specimens were collected from imaged sites (n = 375), and a consensus diagnosis was provided by 2 expert gastrointestinal pathologists and used as the standard. RESULTS: Quantitative information from the high-resolution images was used to develop an algorithm to identify high-grade squamous dysplasia or invasive squamous cell cancer, based on histopathology findings. Optimal performance was obtained using the mean nuclear area as the basis for classification, resulting in sensitivities and specificities of 93% and 92% in the training set, 87% and 97% in the test set, and 84% and 95% in an independent validation set, respectively. CONCLUSIONS: High-resolution microendoscopy with quantitative image analysis can aid in the identification of esophageal squamous neoplasia. Use of software-based image guides may overcome issues of training and expertise in low-resource settings, allowing for widespread use of these optical biopsy technologies.