Journal of Biomedical Optics最新文献

Significance: Spectral-domain optical coherence tomography (SD-OCT) has been widely used in clinical ophthalmic imaging for high spatial resolution and phase stability. The implementation of multiple spectrometers could help resolve the challenges of SD-OCT, including limited imaging speed and sensitivity. However, these two improvements cannot be achieved concurrently.

Aim: We propose a deep-learning-based approach to enhance both imaging speed and sensitivity of SD-OCT systems using a modified U-Net architecture.

Approach: This network adopts a visual state space model to synthesize the high signal-to-noise ratio (SNR) OCT and OCTA from high-speed acquisitions, which bypasses the hardware restriction. The model performance is evaluated both qualitatively and quantitatively using the multiscale structural similarity index measure (MS-SSIM) and contrast-to-noise ratio (CNR).

Results: The results demonstrate effective performance in high-SNR OCT/OCTA reconstruction, providing better contrast between the retinal layers and improved delineation of layer boundaries. Fine structures in both the inner and outer retina, such as microcapillaries and choroid, are successfully restored.

Conclusions: We proposed an effective approach to improve the OCT image quality while maintaining the high acquisition speed using the DNN-based architecture, enabling simultaneous benefits of high imaging speed and enhanced sensitivity.

Significance: Port-wine stains (PWSs) are congenital capillary malformations with the incidence of in newborns of $\sim 0.8\%$ to 2.1%. Hematoporphyrin monomethyl ether-mediated photodynamic therapy (HMME-PDT) has been widely applied in China for PWS. However, there remains substantial room for improvement in both the phototherapeutic selectivity coefficient (PSC) and pain management.

Aim: We investigated the feasibility of modulating transcutaneous oxygen delivery during photodynamic therapy of PWS to enhance therapeutic efficacy and reduce pain.

Approach: A three-dimensional (3D) computational biophysical model was employed to elucidate the mechanisms through which transcutaneous oxygen modulation enhances the therapeutic efficacy of HMME-PDT and improves pain management. The model was constructed to simulate the light propagation, photosensitizer kinetics, oxygen diffusion, and reactive oxygen species (ROS) generation. A treatment optimization strategy based on epidermal oxygen regulation was proposed and evaluated in computational studies. The spatiotemporal distributions of singlet oxygen under normoxic, hypoxic, and anoxic conditions were evaluated, and their effects on treatment-induced pain and lesion-targeted cytotoxicity were analyzed.

Results: Computational analysis showed that compared with normoxic conditions, hypoxia and anoxia significantly enhanced PSC, with improvements of 48% and 61%, respectively. Furthermore, these oxygen-modulated regimens attenuated treatment-associated pain, reducing photochemical pain duration of 17% (hypoxia) and 30% (anoxia). Choosing the right combination of light source irradiance and surface oxygen supply rate amplified therapeutic performance and patient comfort, achieving a 213% increase in PSC and a 57% reduction in photochemical pain duration. These findings establish a mechanistic framework for advancing precision PDT protocols with minimized iatrogenic discomfort.

Conclusions: Established in this computational study, strategic epidermal oxygen restriction critically augments PDT PSC while improving patient tolerance. Computational modeling demonstrates that controlled epidermal hypoxia spatially redistributes oxygen gradients, thereby suppressing superficial ROS generation in nontargeted epidermal layers and selectively concentrating ROS within PWS vasculature. This dual mechanism-simultaneously enhancing therapeutic precision and attenuating treatment-induced pain-presents a pioneering strategy centered on an active oxygen control strategy for enhancing HMME-PDT clinical outcomes. Future research will progress from preclinical validation in animal models to clinical studies to evaluate the therapeutic efficacy and translational potential of this strategy.

Significance: Brillouin spectroscopy noninvasively probes mechanical properties of biological materials and has become a valuable tool in various areas of biomedical research. However, the limited contrast of conventional single stage virtually imaged phased array (VIPA) spectrometers restricts many applications. Current contrast enhancement methods have limitations, including incompatibility with line-scanning collection, which is highly advantageous for fast biomechanical mapping.

Aim: The aim is to develop and characterize a high-throughput contrast enhancement technique using a VIPA-etalon cascade, designed for compatibility with both confocal and line-scanning geometries across a wide range of wavelengths.

Approach: One or more etalons with approximately matched thicknesses are integrated downstream of the VIPA to suppress its Lorentzian tails. A theoretical model and ray-tracing simulation were developed, complemented by experimental validation in confocal and line-scanning setups at 785 and 532 nm, respectively. Brillouin spectra were measured for reference materials and biological samples, including a porcine crystalline lens.

Results: Experimentally, the integration of a single high-finesse cascade yielded contrast enhancements of 27 and $\sim 19\mathrm{dB}$ for the confocal and line-scanning geometries, respectively, with less than 50% reduction in peak transmission in both cases. Simulations demonstrated that substantially higher contrast can be achieved in principle with negligible impact on throughput by serializing multiple low-finesse etalons. High-quality Brillouin spectra were obtained in both collection geometries, demonstrating robust performance across diverse samples.

Conclusions: The VIPA-etalon cascade markedly improves the performance of Brillouin spectroscopy, offering a straightforward, cost-effective, and versatile solution for analyzing biological samples. It enables precise, high-resolution biomechanical investigations, advancing applications in biomedical research and clinical diagnostics.

Significance: Metastasis is a leading cause of cancer-related deaths. Disseminated circulating tumor cells (CTCs) through the bloodstream seed metastatic tumors at distant sites. Most methods for enumerating CTCs in humans clinically rely on drawing and analyzing small blood samples, but these may yield inaccurate estimates of CTC burden and cannot measure CTC changes over time. Identification and enumeration of CTCs for experimental or clinical purposes largely rely on marker-driven analyses by flow cytometry.

Aim: In principle, noninvasive fluorescence enumeration of CTCs directly in vivo could provide a more accurate method for enumerating CTCs. However, this will require a specific contrast agent for CTCs. The goal of this work is to define characteristics of useful CTC contrast agents and perform preliminary testing of candidate contrast agents used for fluorescence-guided surgery (FGS).

Approach: We evaluated a clinical small-molecule folate receptor-targeted molecular imaging agent (OTL38, pafolacianine), a fluorogenic pan-cathepsin imaging agent (VGT-309, abenacianine), and a set of custom-designed, small-molecule prostate-specific membrane antigen (PSMA)-targeted fluorophores. We tested these contrast agents using in vitro cell culture models and in in vivo murine models.

Results: All tested contrast agents showed not only high uptake and labeling by target cell lines but also small but significant labeling of non-cancer blood cells. Contrast agents that exhibited rapid clearance from circulation and the fluorogenic approach resulted in significantly reduced non-specific interfering background fluorescence signals.

Conclusions: Although all of the tested targeted fluorescence contrast agents have properties useful for labeling of CTCs, thus far, none has exhibited the required high specificity. This resulted in some labeling of non-cancer blood cells, which presented false-positive CTC counts. Improved contrast agent design and multiplexed use of more than one contrast agent may improve this specificity.

Significance: Speckle contrast optical spectroscopy (SCOS) and speckle plethysmography (SPG) are increasingly used to measure deep tissue blood flow from tissues. However these methods derive flow from measurements at a single exposure duration, which could introduce errors due to inefficient choice of a single integration time, changes in speckle averaging factors ( $\beta$ ), and noise.

Aim: The aims are to compare and evaluate the robustness of single- and multi-exposure speckle contrast methods in estimating blood flow changes under experimental conditions such as $\beta$ mismatch, noise, and pulsatile flow.

Approach: Speckle visibility was simulated at 10,000 logarithmically spaced exposure durations (0.01 to 10 ms), incorporating $\pm 5$ to 15% noise, using a semi-infinite diffusion model at different physiologically relevant steady state and pulsatile flow rates. Speckle visibility was used to estimate blood flow changes using both the multi-exposure approach (non-linear fitting of the full curve) and conventional single exposure approach at 0.1, 1, and 5 ms durations (SCOS, SPG, and look-up-table), under different noise conditions and mismatches in $\beta$ .

Results: Multi-exposure speckle imaging (MESI) maintained $<1\%$ mean error despite $\beta$ mismatch or noise while maintaining $\le 12\%$ pulsatile-flow error at $\pm 5\%$ noise. Single-exposure methods showed errors up to 90% for $\beta$ mismatch/noise and $\ge 22\%$ pulsatile-flow errors.

Conclusions: MESI outperformed single-exposure approaches in estimating blood flow changes by reducing $\beta$ bias, minimizing noise sensitivity, and accurately tracking pulsatile dynamics.

Significance: Freehand optical ultrasound (OpUS) is an emerging imaging modality that utilizes arrays of fiber-optic, photoacoustical ultrasound sources, and a single fiber-optic detector to perform pulse-echo ultrasound imaging in real time and at video rate. The freehand OpUS imaging probes presented to date have utilized either flat-cleaved optical fibers to generate an array of small, circular sources, or featured an additional array of eccentric optical waveguides to generate elliptical sources that improve ultrasound directivity. However, the incorporation of waveguides imposed severe restrictions on the size of the sources, the source pitch within the array, and the overall size of the imaging probe, and ultimately limited achievable image quality and clinical utility of freehand OpUS probes.

Aim: We present an alternative method for generating eccentric fiber-optic OpUS sources, which incorporates an anisotropic holographic diffuser element (HDE) to shape the profile of fiber-delivered excitation light.

Approach: A commercially available HDE was selected and imprinted into a UV-curable adhesive to improve optical resilience. The monolithic and near-uniform structure of the resulting adhesive-imprinted HDE structure greatly facilitated alignment with, and allowed for arbitrary positioning of, arrays of optical fibers, without adding significant bulk.

Results: The use of an HDE enabled dense, cladding-to-cladding packing of optical fibers, resulting in the first freehand OpUS probe featuring an acoustic source pitch below the spatial Nyquist limit (thus eliminating grating lobe artifacts), high channel count (144 sources), and endoluminal-scale physical dimensions (diameter: 18.5 mm).

Conclusion: This HDE-enabled freehand OpUS imaging probe achieved video-rate, real-time imaging at increased image quality and depth and allowed for the first endoluminal freehand OpUS imaging within a realistic imaging phantom.

Significance: Bacterial contamination poses significant risks, particularly in medical settings, where timely identification is crucial for effective treatment. Traditional diagnostic methods often fail to provide rapid results, underlining the need for alternative approaches.

Aim: We investigate the potential of fluorescence lifetime imaging microscopy (FLIM) for fast label-free classification of bacterial species based on their growth states and intrinsic fluorescence characteristics.

Approach: Utilizing two-photon excitation microscopy, we examined the fluorescence lifetimes of the endogenous fluorophores NAD(P)H and FAD in Escherichia coli K12, Staphylococcus aureus, and Pseudomonas fluorescens. Cells were studied in different growth states-exponential, cooled, and dead-and measurements were taken at excitation wavelengths of 740 and 900 nm. The FLIM data were processed using a multi-component exponential decay model and visualized using phasor plots and kernel density estimation.

Results: Results revealed distinct fluorescence lifetime profiles for each species, with clear differences observed in the exponential growth phase. Notably, E. coli K12 and S. aureus could be distinguished in mixed samples using a single excitation wavelength and emission channel.

Conclusion: These findings demonstrate that FLIM-based metabolic imaging can distinguish between bacterial species without labels, providing a promising basis for rapid, high-resolution diagnostic tools in clinical and research settings. We underscore the potential of FLIM as a powerful tool for bacterial classification, offering advantages in speed and spatial resolution over traditional methods.

Significance: Understanding microbial growth and morphology at the single-cell level is essential for studying microbial physiology, providing valuable insights for research and biotechnology. However, existing workflows often rely on manual cell annotation, which limits efficiency and scalability. Therefore, developing an automated workflow for quantitative analysis of cell growth and morphology is highly desirable.

Aim: We aim to develop an AI-driven analysis system that efficiently segments and indexes microbial cells, and quantitatively analyzes individual cellular features without requiring expensive annotations, for automated monitoring of cell counts and morphological characteristics.

Approach: The automated system consists of four modular components: denoising, zero-shot segmentation using the Segment Anything Model (SAM), structured post-processing, and a quantitative feature extraction. To evaluate the effectiveness of each component, we conducted ablation experiments and systematically studied their impact on the overall system performance.

Results: Denoising and post-processing improved segmentation accuracy by 12.10% and 2.30%, respectively. Among the evaluated SAM variants, the SAM-H model achieved the best performance, with an average error rate of only 3.0% across 1162 manually annotated Escherichia coli cells. Using the optimized SAM-H pipeline, the system efficiently extracted morphometric and intensity features from Escherichia coli cells and nuclei of the yeast and cancer cell lines.

Conclusions: This framework automates quantitative analysis of microbial cells in high-resolution microscopy images. It will enable advanced research on microbial adaptations, with the potential to accelerate studies of extremophiles under harsh environments.

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: 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.