Journal of Biomedical Optics最新文献

Significance: Traditional optical-property image reconstruction techniques are often constrained by artifacts arising from suboptimal source-detector configurations, the amplification of measurement noise during inversion, and limited depth sensitivity, which particularly impacts the accurate reconstruction of deep-seated anomalies such as tumors.

Aim: To overcome these challenges, this research proposes and implements an end-to-end deep learning framework, Channel Attention Fusion Network (CAFNet).

Approach: CAFNet employs AUTOMAP for domain transformation, feature extraction modules for multi-scale feature learning, and channel attention mechanisms to prioritize critical features. The proposed model is trained and tested on simulated and experimental datasets, utilizing metrics such as mean squared error (MSE), peak signal-to-noise ratio (PSNR), and structural similarity index (SSIM) for evaluating model performance.

Results: CAFNet outperforms traditional and state-of-the-art models, achieving the highest SSIM and PSNR values with the lowest MSE. It effectively reconstructs optical properties with high precision, showcasing its ability to detect and localize inclusions in experimental phantoms. An ablation study is performed to highlight the importance of channel attention in CAFNet.

Conclusions: CAFNet demonstrates a significant advancement in diffuse optical imaging, addressing challenges with noise and domain variability issues. Its robust performance highlights the potential in practical medical imaging applications, offering a reliable solution for reconstructing optical properties in complex scenarios.

Significance: Vascular abnormalities may contribute to amyloid-beta accumulation and neurotoxicity in Alzheimer's disease (AD). Monitoring vascular degeneration as AD progresses is essential. Three-photon fluorescence microscopy enables high-resolution deep tissue imaging with minimal invasiveness and photodamage.

Aim: In this proof-of-concept study, we established a longitudinal 3P imaging pipeline to quantify vascular and amyloid plaque changes in the ${\mathrm{APP}}^{\mathrm{NL}\text{-}G\text{-}F}$ mouse model.

Approach: A cranial window allowed repeated 3P imaging at 4-week intervals beginning at 5 weeks after surgery. Vessels labeled with Texas-Red were segmented using DeepVess, whereas plaques labeled with methoxy-XO4 were segmented using custom scripts. Quantitative analyses assessed vascular parameters (diameter, tortuosity, length, inter-vessel distance, total volume) and plaque metrics (radius, total volume).

Results: We imaged the same field over 4 weeks, quantifying an overall decrease in vasculature and an increase in amyloid plaques between two sessions. Significant changes in vessel diameter, inter-vessel distance, and alterations in vessel length and plaque radius were observed. Changes in vessel tortuosity were not significant.

Conclusions: We demonstrate the potential of three-photon imaging to track vascular and amyloid-related changes in deep cortical structures. It offers a tool for studying the interplay between vascular and amyloid pathologies in AD, supporting future research into disease mechanisms and therapeutic strategies.

Significance: Accurate transfer of annotations from histological images to fluorescence images is essential in developing deep learning (DL)-based optical imaging systems for intraoperative assessment of tumor margins. Manual annotation is time-consuming, prone to interobserver variability, and impractical for large-scale datasets.

Aim: We present a semi-automated method that can effectively transfer tumor annotations from pathologist-annotated hematoxylin and eosin (H&E) images to fluorescence images captured using microscopy with ultraviolet surface excitation (MUSE). This method is not intended for intraoperative use but rather to facilitate the creation of annotated datasets for DL model development.

Approach: Our semi-automated method consists of nonrigid image registration, outline extraction and refinement, and annotation transfer. The method was applied to H&E and MUSE image pairs from 35 breast and lung tissue samples. Manual annotations in MUSE images were used as the ground truth for evaluation.

Results: The proposed method achieved a Dice score coefficient of $0.87\pm 0.07$ , convolutional-neural-network-based feature similarity of $0.94\pm 0.04$ , and a normalized Hausdorff distance of $0.15\pm 0.06$ across the dataset.

Conclusion: These results demonstrate that the method provides a fast and accurate solution for generating annotated MUSE datasets necessary for training DL algorithms for intraoperative tumor margin detection.

Significance: The lymphatic system, crucial for immune function and fluid balance, is difficult to study due to its nearly invisible channels and passive movements. Despite its importance, real-time, noninvasive lymphatic imaging is limited and often relies on exogenous agents. A largely overlooked feature of the lymphatic system is its natural hypoxia, arising because lymphatic vessels and nodes are distant from oxygen-rich blood and serve as a waste reservoir and from amplifying factors such as inflammation. This hypoxia has been linked to lymphangiogenesis and cancer metastasis.

Aim: We introduce a method for real-time macroscopic imaging of lymphatic function and response to inflammation, through the intrinsic hypoxia transients that occur in lymphatic structures.

Approach: The naturally hypoxic environment of the lymphatic system was imaged in vivo in mice through the delayed fluorescence (DF) of metabolized endogenous protoporphyrin IX (PpIX), induced by 5-aminolevulinic acid. PpIX localizes to inflamed regions; when inflammation is cleared by lymphatics, the low oxygen conditions cause the DF signal to be amplified. DF imaging and lymphatic kinetics were characterized in wound, inflammation, and pancreatic tumor models. High-intensity popliteal and sentinel nodes, wounds, and tumors were excised for immunohistochemistry (IHC) and hematoxylin and eosin analysis.

Results: Lymphatic pumping frequency changed with increasing wound severity, and hypoxia appeared in sentinel nodes near tumors. Cyclical pumping occurred at edema sites and in wound and tumor-adjacent nodes. Uninjured anesthetized mice showed little contrast, whereas awake mice exhibited hypoxia localized to lymph nodes. Microscopy and IHC confirmed PpIX and hypoxia presence in nodes, tumors, and wounds, localized to macrophages and T cells.

Conclusions: Unlike injection-based regional lymph node mapping, DF hypoxia imaging appears to provide a natural whole-body contrast mechanism, highlighting its potential for visualizing lymphatic function and associated hypoxia dynamics. This original documentation of lymphatic hypoxia has potential applications in surgical guidance, tracking of metastatic tumors, and immune response tracking.

Significance: Collagen, the main load-bearing component in tissue, is present in all animals and forms a variety of networks from the fibrils, fibers, bundles, and lamellae into which it self-assembles. The collagen microstructure is different among tissue types, and the different microstructures give rise to tissue-specific mechanical properties. Therefore, methods for visualizing collagen fibers and their orientation are essential for understanding the biomechanical properties of tissue.

Aim: Our aim in this review is to provide the basis for understanding the methodology of polarized light imaging methods and how they can be used to characterize collagen microstructure.

Approach: We begin with a description of collagen microstructure and its relationship to tissue biomechanics, a basic formalism of polarized light, and how collagen interacts with polarized light. We then describe polarized light microscopy and its various forms, particularly instant polarized light microscopy, then polarization-sensitive optical coherence tomography, and last, polarization-resolved second-harmonic generation microscopy.

Results: We describe methods for imaging collagen microstructure with polarized light from in vivo methods to high-resolution volumetric imaging of tissue sections.

Conclusions: We intend to help those interested in using polarized light to image and understand the relationship between collagen microstructure and biomechanics.

Significance: Short-wave infrared (SWIR) light has recently gained popularity in tissue spectroscopy and imaging applications for a wide range of biomedical applications, primarily due to advancements in hardware (e.g., cameras).

Aim: We aim to provide a detailed review of SWIR-based biomedical optics studies from the past decade, during which there has been a proliferation of SWIR-based tissue-optics studies.

Approach: We report literature occurring after the publication of our previous (2015) review of this space, describing next-generation SWIR-based techniques that hold significant promise for enhanced in vivo tissue characterization and clinical translation.

Results: Interest from the biophotonics field in SWIR technology is typically attributable to (1) the capability of SWIR light to provide greater sensitivity to chromophores such as water and lipids, with absorption peaks not as prominent in the visible-to-near-infrared (VIS-NIR) spectral region, and (2) the potential for SWIR photons to penetrate through superficial tissue layers due to lower scattering in the SWIR than in the VIS-NIR, as well as substantially reduced attenuation from hemoglobin and melanin.

Conclusion: This review of emerging SWIR biophotonic technologies illustrates the rapid growth in the use of SWIR light for in vivo tissue spectroscopy and imaging.

Significance: Integrating ultrasound (US) with multiphoton microscopy (MPM) enables comprehensive tissue characterization by combining contrast mechanisms across spatial scales. However, prior implementations suffer from degraded US image quality at the glass optical window arising from off-axis acoustic noise (clutter).

Aim: We aimed to reduce clutter near the optical window without compromising the MPM image quality using different optical window materials combined with different ultrasound beamforming techniques.

Approach: Clutter in B-mode images was measured for glass and several optically clear polymer films using physically or synthetically focused US beamforming with varied focal configurations ( $f/\#$ ) and with acoustic coupling beneath the window. MPM image quality with the material optical windows was assessed via second harmonic generation (SHG) point spread function (PSF) measurements. We evaluated SHG-based automated collagen fiber alignment quantification with the optical windows in rat tail tendon samples and collagen gels.

Results: Candidate materials included polymethyl pentene (PMP), cyclin olefin copolymer (COC), polycarbonate (PC), polyethylene terephthalate, polymethyl methacrylate, and an Ibidi^® polymer coverslip. At receive $f/\#\ge 2$ , COC and Ibidi^® reduced acoustic clutter by $>40\%$ compared with glass with physical focusing regardless of physically or synthetically focused beams or acoustic coupling. Collagen gel imaged with COC and high $f/\#$ showed clearer ultrasound speckle close to the optical windows. COC, Ibidi^®, PMP, and PC showed minimal SHG PSF differences from glass and collagen alignment errors $<5\%$ relative to glass.

Conclusions: COC or Ibidi^® optical windows significantly improve US image quality without compromising MPM quality. These enhancements support future multimodal studies with high-quality, co-registered US and MPM data.

Significance: Colorectal cancer (CRC) remains a major cause of cancer-related mortality, with incomplete resection contributing to recurrence and poor survival. Improving intraoperative margin detection is critical to enhance surgical precision and patient outcomes.

Aim: We aim to evaluate the tumor-targeting specificity of cetuximab-IRDye800CW (Cet-IRDye800CW), an epidermal growth factor receptor (EGFR)-specific near-infrared probe, for fluorescence-guided surgery in CRC models.

Approach: Cet-IRDye800CW and IgG-IRDye800CW controls were synthesized and tested in LS174T and SW948 xenografts as well as patient-derived xenografts (PDXs). Mice received $200\mu g$ of the probe intravenously and underwent daily fluorescence imaging for 10 days. Tumor-to-background ratio (TBR) and mean fluorescence intensity (MFI) were quantified, and EGFR expression was analyzed by Western blot, reverse transcription quantitative polymerase chain reaction, and immunohistochemistry.

Results: Cetuximab-IRDye800CW significantly enhanced tumor fluorescence compared with controls in all models ( $p<0.05$ ). TBR increased progressively and peaked on day 10 (LS174T: $25.6\pm 2.94$ ; SW948: $21.6\pm 1.71$ ), whereas background MFI declined, resulting in improved tumor contrast. Tumor-specific fluorescence was detectable from day 1 and intensified over time. Similar findings were observed in PDX models, consistent with high EGFR expression.

Conclusion: Cetuximab-IRDye800CW provides stable, tumor-specific, high-contrast fluorescence imaging in EGFR-high CRC models. These findings validate its molecular targeting capability and support its translational potential for fluorescence-guided CRC surgery.

Significance: High-throughput live-cell imaging is crucial for biological applications, including organ-on-a-chip (OoaC) platforms, yet conventional optical systems face a fundamental trade-off between magnification and field of view (FOV). This limitation hinders the ability to capture large-scale biological dynamics while maintaining single-cell resolution. We address this gap by introducing a scalable, high-resolution imaging solution specifically tailored for OoaC platforms and other microfluidic-based systems.

Aim: We aim to develop a dual-mode multichannel optical imaging system capable of achieving single-cell resolution over an extended FOV while maintaining a working distance suitable for integration with microfluidic devices.

Approach: The system employs microlens arrays in conjunction with laser-fabricated micro-aperture arrays to optically isolate imaging channels, minimizing crosstalk. Two operational modes are implemented: (1) rapid sampling mode for instantaneous, partial-area imaging and (2) full-field imaging mode, utilizing micro-scanning and computational stitching to generate a seamless high-resolution composite. The system's performance was validated through experimental imaging and theoretical modeling.

Results: The system achieves an FOV of $8.4\times 6m{m}^2$ at 4× magnification with single-cell resolution while preserving a 14 mm working distance. Experimental results closely align with theoretical expectations, confirming high-fidelity imaging without requiring a large sensor. Dual-mode functionality enables both rapid assessments and detailed, large-area imaging, enhancing its applicability in biological research.

Conclusions: This compact and scalable imaging system overcomes the traditional magnification-FOV trade-off, offering a powerful tool for drug screening, cellular dynamics studies, and microfluidic-based biological analyses. Its high-resolution capability and adaptability make it a valuable asset for advancing OoaC technologies.