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Background: Medical devices for fluorescence-guided surgery (FGS) are becoming available at a fast pace. The main challenge for surgeons lies in the lack of in-depth knowledge of optical imaging, different technical specifications and poor standardisation, and the selection of the best device based on clinical application., Methods: This manuscript aims to provide an up-to-date description of the commercially available fluorescence imaging platforms by comparing their mode of use, required settings, image types, compatible fluorophores, regulatory approval, and cost. We obtained this information by performing a broad literature search on PubMed and by contacting medical companies directly. The data for this review were collected up to November 2023., Results: Thirty-two devices made by 19 medical companies were identified. Ten systems are surgical microscopes, 5 can be used for both open and minimally invasive surgery (MIS), 6 can only be used for open surgery, and 10 only for MIS. One is a fluorescence system available for the Da Vinci robot. Nineteen devices can provide an overlay between fluorescence and white light image. All devices are compatible with Indocyanine Green, the most common fluorescence dye used intraoperatively. There is significant variability in the hardware and software of each device, which resulted in different sensitivity, fluorescence intensity, and image quality. All devices are CE-mark regulated, and 30 were FDA-approved., Conclusion: There is a prolific market of devices for FGS and healthcare professionals should have basic knowledge of their technical specifications to use it at best for each clinical indication. Standardisation across devices must be a priority in the field of FGS, and it will enhance external validity for future clinical trials in the field., (© 2024. Crown.)

Disparities in surgical outcomes often result from subjective decisions dictated by surgical training, experience, and available resources. To improve outcomes, surgeons have adopted advancements in robotics, endoscopy, and intra-operative imaging including fluorescence-guided surgery (FGS), which highlights tumors and anatomy in real-time. However, technical, economic, and logistic challenges hinder widespread adoption of FGS beyond high-resource centers. To overcome these impediments, we combined laser diodes, Raspberry Pi cameras and computers, off-the-shelf optical components, and 3D-printed parts to make a battery-powered, compact, dual white light and NIR imaging system that has comparable performance to existing bulkier, pricier, and wall-powered technologies. We combined these components with off-the-shelf augmented reality (AR) glasses to create a fully-wearable fluorescence imaging AR Raspberry Pi-based goggle system (FAR-Pi) and validated performance in a pre-clinical cancer surgery model. Novel device design ensures distance-independent coalignment between real and augmented views. As an open-source, affordable, and adaptable system, FAR-Pi is poised to democratize access to FGS and improve health outcomes worldwide., (© 2024. The Author(s).)

The accumulation of micro/nanoplastics (MNPs) in ecosystems poses tremendous environmental risks for terrestrial and aquatic organisms. Designing rapid, field-deployable, and sensitive devices for assessing the potential risks of MNPs pollution is critical. However, current techniques for MNPs detection have limited effectiveness. Here, we design a wireless portable device that allows rapid, sensitive, and on-site detection of MNPs, followed by remote data processing via machine learning algorithms for quantitative fluorescence imaging. We utilized a supramolecular labeling strategy, employing luminescent metal-phenolic networks composed of zirconium ions, tannic acid, and rhodamine B, to efficiently label various sizes of MNPs (e.g., 50 nm-10 μm). Results showed that our device can quantify MNPs as low as 330 microplastics and 3.08 × 10 6 nanoplastics in less than 20 min. We demonstrated the applicability of the device to real-world samples through determination of MNPs released from plastic cups after hot water and flow induction and nanoplastics in tap water. Moreover, the device is user-friendly and operative by untrained personnel to conduct data processing on the APP remotely. The analytical platform integrating quantitative imaging, customized data processing, decision tree model, and low-cost analysis ($0.015 per assay) has great potential for high-throughput screening of MNPs in agrifood and environmental systems.

Significance: Intraoperative molecular imaging (IMI) enables the detection and visualization of cancer tissue using targeted radioactive or fluorescent tracers. While IMI research has rapidly expanded, including the recent Food and Drug Administration approval of a targeted fluorophore, the limits of detection have not been well-defined., Aim: The ability of widely available handheld intraoperative tools (Neoprobe and SPY-PHI) to measure gamma decay and fluorescence intensity from IMI tracers was assessed while varying characteristics of both the signal source and the intervening tissue or gelatin phantoms., Approach: Gamma decay signal and fluorescence from tracer-bearing tumors (TBTs) and modifiable tumor-like inclusions (TLIs) were measured through increasing thicknesses of porcine tissue and gelatin in custom 3D-printed molds. TBTs buried beneath porcine tissue were used to simulate IMI-guided tumor resection., Results: Gamma decay from TBTs and TLIs was detected through significantly thicker tissue and gelatin than fluorescence, with at least 5% of the maximum signal observed through up to 5 and 0.5 cm, respectively, depending on the overlying tissue type or gelatin., Conclusions: We developed novel systems that can be fine-tuned to simulate variable tumor characteristics and tissue environments. These were used to evaluate the detection of fluorescent and gamma signals from IMI tracers and simulate IMI surgery., (© 2024 The Authors.)

Background: Tomato quality visual grading is greatly affected by the problems of smooth skin, uneven illumination and invisible defects that are difficult to identify. The realization of intelligent detection of postharvest epidermal defects is conducive to further improving the economic value of postharvest tomatoes., Results: An image acquisition device that utilizes fluorescence technology has been designed to capture a dataset of tomato skin defects, encompassing categories such as rot defects, crack defects and imperceptible defects. The YOLOv5m model was improved by introducing Convolutional Block Attention Module and replacing part of the convolution kernels in the backbone network with Switchable Atrous Convolution. The results of comparison experiments and ablation experiments show that the Precision, Recall and mean Average Precision of the improved YOLOv5m model were 89.93%, 82.33% and 87.57%, which are higher than YOLOv5m, Faster R-CNN and YOLOv7, and the average detection time was reduced by 47.04 ms picture -1 ., Conclusion: The present study utilizes fluorescence imaging and an improved YOLOv5m model to detect tomato epidermal defects, resulting in better identification of imperceptible defects and detection of multiple categories of defects. This provides strong technical support for intelligent detection and quality grading of tomatoes. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Freehand optical ultrasound (OpUS) imaging is an emerging ultrasound imaging paradigm that uses an array of fibre-optic, photoacoustic ultrasound sources and a single fibre-optic ultrasound detector to perform ultrasound imaging without the need for electrical components in the probe head. Previous freehand OpUS devices have demonstrated capability for real-time, video-rate imaging of clinically relevant targets, but have been hampered by poor ultrasound penetration, significant imaging artefacts and low frame rates, and their designs limited their clinical applicability. In this work we present a novel freehand OpUS imaging platform, including a fully mobile and compact acquisition console and an improved probe design. The novel freehand OpUS probe presented utilises optical waveguides to shape the generated ultrasound fields for improved ultrasound penetration depths, an extended fibre-optic bundle to improve system versatility and an overall ruggedised design with protective elements to improve probe handling and protect the internal optical components. This probe is demonstrated with phantoms and the first multi-participant in vivo imaging study conducted with freehand OpUS imaging probes, this represents several significant steps towards the clinical translation of freehand OpUS imaging., (© 2024. The Author(s).)

Significance: Spatial frequency domain imaging (SFDI) applies patterned near-infrared illumination to quantify the optical properties of subsurface tissue. The periocular region is unique due to its complex ocular adnexal anatomy. Although SFDI has been successfully applied to relatively flat in vivo tissues, regions that have significant height variations and curvature may result in optical property inaccuracies., Aim: We characterize the geometric impact of the periocular region on SFDI imaging reliability., Approach: SFDI was employed to measure the reduced scattering coefficient ( μ s ' ) and absorption coefficient ( μ a ) of the periocular region in a cast facial tissue-simulating phantom by capturing images along regions of interest (ROIs): inferior temporal quadrant (ITQ), inferior nasal quadrant (INQ), superior temporal quadrant (STQ), central eyelid margin (CEM), rostral lateral nasal bridge (RLNB), and forehead (FH). The phantom was placed on a chin rest and imaged nine times from an "en face" or "side profile" position, and the flat back of the phantom was measured 15 times., Results: The measured μ a and μ s ' of a cast facial phantom are accurate when comparing the ITQ, INQ, STQ, and FH to its flat posterior surface. Paired t tests of ITQ, INQ, STQ, and FH μ a and μ s ' concluded that there is not enough evidence to suggest that imaging orientation impacted the measurement accuracy. Regions of extreme topographical variation, i.e., CEM and RLNB, did exhibit differences in measured optical properties., Conclusions: We are the first to evaluate the geometric implications of wide-field imaging along the periocular region using a solid tissue-simulating facial phantom. Results suggest that the ITQ, INQ, STQ, and FH of a generalized face have minimal impact on the SFDI measurement accuracy. Areas with heightened topographic variation exhibit measurement variability. Device and facial positioning do not appear to bias measurements. These findings confirm the need to carefully select ROIs when measuring optical properties along the periocular region., (© 2024 The Authors.)

Optical methods based on thin multimode fibers (MMFs) are promising tools for measuring neuronal activity in deep brain regions of freely moving mice thanks to their small diameter. However, current methods are limited: while fiber photometry provides only ensemble activity, imaging techniques using of long multimode fibers are very sensitive to bending and have not been applied to unrestrained rodents yet. Here, we demonstrate the fundamentals of a new approach using a short MMF coupled to a miniscope. In proof-of-principle in vitro experiments, we disentangled spatio-temporal fluorescence signals from multiple fluorescent sources transmitted by a thin (200 µm) and short (8 mm) MMF, using a general unconstrained non-negative matrix factorization algorithm directly on the raw video data. Furthermore, we show that low-cost open-source miniscopes have sufficient sensitivity to image the same fluorescence patterns seen in our proof-of-principle experiment, suggesting a new avenue for novel minimally invasive deep brain studies using multimode fibers in freely behaving mice., (© 2024. The Author(s).)

Significance: Single-chip imaging devices featuring vertically stacked photodiodes and pixelated spectral filters are advancing multi-dye imaging methods for cancer surgeries, though this innovation comes with a compromise in spatial resolution. To mitigate this drawback, we developed a deep convolutional neural network (CNN) aimed at demosaicing the color and near-infrared (NIR) channels, with its performance validated on both pre-clinical and clinical datasets., Aim: We introduce an optimized deep CNN designed for demosaicing both color and NIR images obtained using a hexachromatic imaging sensor., Approach: A residual CNN was fine-tuned and trained on a dataset of color images and subsequently assessed on a series of dual-channel, color, and NIR images to demonstrate its enhanced performance compared with traditional bilinear interpolation., Results: Our optimized CNN for demosaicing color and NIR images achieves a reduction in the mean square error by 37% for color and 40% for NIR, respectively, and enhances the structural dissimilarity index by 37% across both imaging modalities in pre-clinical data. In clinical datasets, the network improves the mean square error by 35% in color images and 42% in NIR images while enhancing the structural dissimilarity index by 39% in both imaging modalities., Conclusions: We showcase enhancements in image resolution for both color and NIR modalities through the use of an optimized CNN tailored for a hexachromatic image sensor. With the ongoing advancements in graphics card computational power, our approach delivers significant improvements in resolution that are feasible for real-time execution in surgical environments., (© 2024 The Authors.)

Background: Autofluorescence photography can detect specific light-tissue interactions and record important pathophysiological changes associated with nonmelanoma skin cancer (NMSC), which has been ascribed to the fluorescence of an aromatic amino acid, tryptophan., Objective: To assess the impact of a novel, autofluorescence imaging (AFI) device on margin control for NMSCs before Mohs micrographic surgery (MMS) in an effort to decrease overall operating time., Methods: Before the initial stage of MMS, NMSCs were measured with a 2-mm margin as standard of care (normal margin). The tumor was then imaged with the AFI device. A 2-mm margin was drawn around the fluorescent area captured by the AFI device and was referred to as the camera margin. The tumor was excised based on the normal margin and evaluated on frozen histological section., Results: Imaging based on the AFI device resulted in appropriate recommendations for margin control in 8 of 11 tumors. Four of these tumors did not fluoresce and demonstrated a lack of tumor residuum on stage I specimen, as anticipated. There were no side effects from the AFI device., Conclusion: This is an initial pilot study that supports the use of a novel, noninvasive imaging device to help with margin assessment before MMS. On optimization, this device has potential to extend applicability to surgical excisions for tumors that do not fulfill criteria for MMS., (Copyright © 2024 by the American Society for Dermatologic Surgery, Inc. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Reproductive management significantly impacts dairy farm productivity, necessitating accurate timely pregnancy detection in cattle. This paper presents a novel handheld and portable fluorescence imaging system designed for quantitative assessment of pregnancy-specific biomarkers, addressing the limitations of current detection methods. The objective was to develop a cost-effective, at-farm solution for detecting pregnancy-specific protein B (PSPB) in bovine plasma samples. The system integrates an imaging module and a custom software application, enabling image capture, data processing, and PSPB concentration determination. Calibration utilizing known PSPB concentrations achieved a 0.6 ng/mL limit of detection. Validation encompassed a comparison with a standard ELISA method using 100 bovine plasma samples; minimal bias and good agreement were observed within the linear range of the calibration curve for both methods. The system offers portability, user-friendliness, and potential for multiplex detection, promising real-time, at-farm reproductive management. This study demonstrates the successful development and validation of a portable fluorescence imaging system, offering an efficient and accurate approach to detecting pregnancy-specific biomarkers in cattle. Its implications extend to improving dairy farm productivity by enabling timely and reliable reproductive management practices., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Objective. Effective treatment within 3-5 months of disease onset significantly improves rheumatoid arthritis (RA) prognosis. Nevertheless, 30% of RA patients fail their first treatment, and it takes 3-6 months to identify failure with current monitoring techniques. Time-domain diffuse optical imaging (TD-DOI) may be more sensitive to RA disease activity and could be used to detect treatment failure. In this report, we present the development of a TD-DOI hand imaging system and validate its ability to measure simulated changes in RA disease activity using tissue-mimicking finger phantoms. Approach. A TD-DOI system was built, based on a single-pixel camera architecture, and used to image solid phantoms which mimicked a proximal interphalangeal finger joint. For reference, in silico images of virtual models of the solid phantoms were also generated using Monte Carlo simulations. Spatiotemporal Fourier components were extracted from both simulated and experimental images, and their ability to distinguish between phantoms representing different RA disease activity was quantified. Main results. Many spatiotemporal Fourier components extracted from TD-DOI images could clearly distinguish between phantoms representing different states of RA disease activity. Significance. A TD-DOI system was built and validated using finger-mimicking solid phantoms. The findings suggest that the system could be used to monitor RA disease activity. This single-pixel TD-DOI system could be used to acquire longitudinal measures of RA disease activity to detect early treatment failure., (Creative Commons Attribution license.)

Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition., Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells., Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto , three-dimensional, high-resolution visualization of cells cultured on microcarriers., Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography., Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes., (© 2024 The Authors.)

Time-resolved fluorescence imaging techniques, like confocal fluorescence lifetime imaging microscopy, are powerful photonic instrumentation tools of modern science with diverse applications, including: biology, medicine, and chemistry. However, complexities of the systems, both at specimen and device levels, cause difficulties in quantifying soft biomarkers. To address the problems, we first aim to understand and model the underlying photophysics of fluorescence decay curves. For this purpose, we provide a set of mathematical functions, called "life models", fittable with the real temporal recordings of histogram of photon counts. For each model, an equivalent electrical circuit, called a "life circuit", is derived for explaining the whole process. In confocal endomicroscopy, the components of excitation laser, specimen, and fluorescence-emission signal as the histogram of photon counts are modelled by a power source, network of resistor-inductor-capacitor circuitry, and multimetre, respectively. We then design a novel pixel-level temporal classification algorithm, called a "fit-flexible approach", where qualities of "intensity", "fall-time", and "life profile" are identified for each point. A model selection mechanism is used at each pixel to flexibly choose the best representative life model based on a proposed Misfit-percent metric. A two-dimensional arrangement of the quantified information detects some kind of structural information. This approach showed a potential of separating microbeads from lung tissue, distinguishing the tri-sensing from conventional methods. We alleviated by 7% the error of the Misfit-percent for recovering the histograms on real samples than the best state-of-the-art competitor. Codes are available online.

To translate near-infrared (NIR) and shortwave infrared (SWIR) fluorescence imaging into the clinic, the paired imaging device needs to detect trace doses of fluorescent imaging agents. Except for the filtration scheme and excitation light source, the image sensor used will finally determine the detection limitations of NIR and SWIR fluorescence imaging systems. In this review, we investigate the current state-of-the-art image sensors used in NIR and SWIR fluorescence imaging systems and discuss the advantages and limitations of their characteristics, such as readout architecture and noise factors. Finally, the imaging performance of these image sensors is evaluated and compared.

Fluorescence microscopy has undergone rapid advancements, offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However, the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here, we introduce sHAPR, a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings, enabling acquisition at the maximum camera readout rate, typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems, including high-throughput flow cytometry, cardiomyocyte contraction, and neuronal calcium waves, using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities, opening up new avenues for investigating high-speed biological phenomena., (© 2024. The Author(s).)

Advanced glycation end-products (AGEs) are complex compounds closely associated with several chronic diseases, especially diabetes mellitus (DM). Current methods for detecting AGEs are not suitable for screening large populations, or for long-term monitoring. This paper introduces a portable autofluorescence detection system that measures the concentration of AGEs in the skin based on the fluorescence characteristics of AGEs in biological tissues. The system employs a 395 nm laser LED to excite the fluorescence of AGEs, and uses a photodetector to capture the fluorescence intensity. A model correlating fluorescence intensity with AGEs concentration facilitates the detection of AGEs levels. To account for the variation in optical properties of different individuals' skin, the system includes a 520 nm light source for calibration. The system features a compact design, measuring only 60 mm × 50 mm × 20 mm, and is equipped with a miniature STM32 module for control and a battery for extended operation, making it easy for subjects to wear. To validate the system's effectiveness, it was tested on 14 volunteers to examine the correlation between AGEs and glycated hemoglobin, revealing a correlation coefficient of 0.49. Additionally, long-term monitoring of AGEs' fluorescence and blood sugar levels showed a correlation trend exceeding 0.95, indicating that AGEs reflect changes in blood sugar levels to some extent. Further, by constructing a multivariate predictive model, the study also found that AGEs levels are correlated with age, BMI, gender, and a physical activity index, providing new insights for predicting AGEs content and blood sugar levels. This research supports the early diagnosis and treatment of chronic diseases such as diabetes, and offers a potentially useful tool for future clinical applications.

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Franz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Cherenkov luminescence imaging has shown potential for relative dose distribution and field verification in radiation therapy. However, to date, limited research utilizing Cherenkov luminescence for absolute dose calibration has been conducted owing to uncertainties arising from camera positioning and tissue surface optical properties., Purpose: This paper introduces a novel approach to multispectral Cherenkov luminescence imaging combined with Fricke-xylenol orange gel (FXG) film, termed MCIFF, which can enable online full-field absolute dose measurement. By integrating these two approaches, MCIFF allows for calibration of the ratio between two spectral intensities with absorbed dose, thereby enabling absolute dose measurement., Methods: All experiments are conducted on a Varian Clinac 23EX, utilizing an electron multiplying charge-coupled device (EMCCD) camera and a two-way image splitter for simultaneous capture of two-spectral Cherenkov imaging. In the first part of this study, the absorbance curves of the prepared FXG film, which receives different doses, are measured using a fluorescence spectrophotometer to verify the correlation between absorbance and dose. In the second part, the FXG film is positioned directly under the radiation beam to corroborate the dose measurement capacity of MCIFF across various beams. In the third part, the feasibility of MCIFF is tested in actual radiotherapy settings via a humanoid model, demonstrating its versatility with various radiotherapy materials., Results: The results of this study indicate that the logarithmic ratios of spectral intensities at wavelengths of 550 ± 50 and 700 ± 100 nm accurately reflect variations in radiation dose (R 2  > 0.96) across different radiation beams, particle energies, and dose rates. The slopes of the fitting lines remain consistent under varying beam conditions, with discrepancies of less than 8%. The optical profiles obtained using the MCIFF exhibit a satisfactory level of agreement with the measured results derived from the treatment planning system (TPS) and EBT3 films. Specifically, for photon beams, the lateral distances between the 80% and 20% isodose lines, referred to as the penumbra (P80-20) values, obtained through TPS, EBT3 films, and MCIFF, are determined as 0.537, 0.664, and 0.848 cm, respectively. Similarly, for electron beams, the P80-20 values obtained through TPS, EBT3 films, and MCIFF are found to be 0.432, 0.561, and 0.634 cm, respectively. Furthermore, imaging of the anthropomorphic phantom demonstrates the practical application of MCIFF in real radiotherapy environments., Conclusion: By combining an FXG film with Cherenkov luminescence imaging, MCIFF can calibrate Cherenkov luminescence to absorbed dose, filling the gap in online 2D absolute dose measurement methods in clinical practice, and providing a new direction for the clinical application of optical imaging to radiation therapy., (© 2024 American Association of Physicists in Medicine.)

Ultrasound switchable fluorescence (USF) imaging, a hybrid imaging technology that combines the advantages of both fluorescence sensitivity and acoustic resolution in centimeter-deep tissue, has great potential for biomedical different applications. A camera-based USF imaging system reveals its capability of capturing both spatial and temporal dynamics of the USF signal in tissue. In this study, various algorithms were explored to enhance the spatial resolution and signal-to-noise ratio (SNR) of USF images, utilizing temporal and spatial information from a camera-based time-domain USF imaging system. The correlation method proved effective in boosting SNR, while the ascending-slope-weighted method enhanced spatial resolution. Additionally, the spatially back-projection method significantly improved spatial resolution in silicone phantoms. The results underscore the advantages of incorporating temporal and spatial information from USF signals., (© 2024 Wiley‐VCH GmbH.)

Background: Tissue mimicking optical phantoms are commonly used to calibrate or validate the performance of near-infrared spectroscopy or tomography. Human tissue is not only irregular in shape, but also exhibits dynamic behaviour, which can cause changes in optical properties. However, existing phantoms lack complex structures and/or continuously varying optical properties., Aim: The project aimed to design, fabricate and characterise a novel phantom system for testing near-infrared imaging devices., Material and Methods: We designed a dynamic tissue-mimicking phantom platform which features arbitrary internal shapes and variable optical properties. The solid part of phantom was made of silicone material with absorbing and scattering properties similar to the brain. We printed a semi-ellipsoidal sphere (a major axis = 20 mm and a minor axis = the third axis = 12 mm) using a water-soluble material polyvinyl alcohol (PVA). The shape was placed at the depth of 5 mm in the silicone bulk. The desired internal hollow structure was formed after curing and submerging the phantom in water. The liquid part contained dyes and Intralipid. The optical properties within the internal shape were adjusted by injecting the liquid solutions of varying dye concentrations with a syringe pump at a constant rate. The phantom was measured by a frequency domain near-infrared spectroscopy (FD NIRS) and imaged by a time domain near-infrared optical tomography (TD NIROT)., Results and Discussion: A dynamic phantom system with a complex internal structure and varying optical properties was created. Changes in light intensity were detected by the FD NIRS. The internal structure of this phantom was accurately recovered by NIROT image reconstruction., Conclusion: We successfully developed a novel phantom system with an internal complex shape and continuously adjustable optical properties. This phantom was accurately imaged using NIROT, and the changing light intensity was detected by NIRS. It is a valuable tool for validating optical technologies., (© 2024. Oxygen Transport to Tissue International.)

Significance: Wavelength selection from a large diffuse reflectance spectroscopy (DRS) dataset enables removal of spectral multicollinearity and thus leads to improved understanding of the feature domain. Feature selection (FS) frameworks are essential to discover the optimal wavelengths for tissue differentiation in DRS-based measurements, which can facilitate the development of compact multispectral optical systems with suitable illumination wavelengths for clinical translation., Aim: The aim was to develop an FS methodology to determine wavelengths with optimal discriminative power for orthopedic applications, while providing the frameworks for adaptation to other clinical scenarios., Approach: An ensemble framework for FS was developed, validated, and compared with frameworks incorporating conventional algorithms, including principal component analysis (PCA), linear discriminant analysis (LDA), and backward interval partial least squares (biPLS)., Results: Via the one-versus-rest binary classification approach, a feature subset of 10 wavelengths was selected from each framework yielding comparable balanced accuracy scores (PCA: 94.8 ± 3.47 % , LDA: 98.2 ± 2.02 % , biPLS: 95.8 ± 3.04 % , and ensemble: 95.8 ± 3.16 % ) to those of using all features (100%) for cortical bone versus the rest class labels. One hundred percent balanced accuracy scores were generated for bone cement versus the rest. Different feature subsets achieving similar outcomes could be identified due to spectral multicollinearity., Conclusions: Wavelength selection frameworks provide a means to explore domain knowledge and discover important contributors to classification in spectroscopy. The ensemble framework generated a model with improved interpretability and preserved physical interpretation, which serves as the basis to determine illumination wavelengths in optical instrumentation design., (© 2023 The Authors.)

In two articles in this issue, Fujiwara et al. developed an ultrasensitive high-speed camera capable of single-molecule fluorescence imaging at a microsecond timescale (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202110160). This major leap in detection speed enables the organization of plasma membrane and integrin-based adhesions to be probed in unprecedented detail (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202110162)., (© 2023 Huang and Kanchanawong.)

The study was aimed to evaluate the performance of a newly developed spectroscopy-based non-invasive and noncontact device (SAMIRA) for the simultaneous measurement of hemoglobin, bilirubin and oxygen saturation as an alternative to the invasive biochemical method of blood sampling. The accuracy of the device was assessed in 4318 neonates having incidences of either anemia, jaundice, or hypoxia. Transcutaneous bilirubin, hemoglobin and blood saturation values were obtained by the newly developed instrument which was corroborated with the biochemical blood tests by expert clinicians. The instrument is trained using Artificial Neural Network Analysis to increase the acceptability of the data. The artificial intelligence incorporated within the instrument determines the disease condition of the neonate. The Pearson's correlation coefficient, r was found to be 0.987 for hemoglobin estimation and 0.988 for bilirubin and blood gas saturation respectively. The bias and the limits of agreement for the measurement of all the three parameters were within the clinically acceptance limit., (© 2023. The Author(s).)

With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with "clickable" side chains into proteins of interest. Well-defined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl-tRNA synthetase (chPylRS 2020 ) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label β-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecular-scale optical three-dimensional resolution.

Background: In selected patients with peritoneal metastases of colorectal origin, complete cytoreduction has been the main single prognostic factor influencing long-term outcomes. In these patients, indocyanine green fluorescence imaging seems to be useful in detecting small subclinical peritoneal implants. However, quantitative fluorescence analysis has not yet been established as standard., Objective: This study aimed to evaluate the sensitivity and specificity of quantitative indocyanine green fluorescence assessment in the detection of peritoneal metastases of nonmucinous colorectal origin., Design: This is a single-center, single-arm, low-intervention prospective trial., Settings: A fluorescence assessment device was used for intraoperative fluorescence quantitative assessment., Patients: Consecutive patients diagnosed with peritoneal metastases of colorectal origin who met the inclusion criteria were selected for curative surgery., Interventions: Intravenous indocyanine green was administered 12 hours before surgery. Cytoreduction was performed through nodule identification under white light and then under indocyanine green. Finally, ex vivo fluorescence was assessed., Main Outcome Measures: The primary outcomes measured were the sensitivity and specificity of quantitative fluorescence., Results: The first 11 enrolled patients were included in this preliminary analysis. In total, 52 nodules were resected, with 37 (71.1%) being diagnosed as malignant in the histopathological analysis. Of those, 5 (13.5%) were undetectable under white light and were identified only with fluorescence. A total of 15 nonmalignant nodules were detected under white light, 8 (53.3%) of which were fluorescence negative. Fluorescence greater than 181 units might be the threshold of malignancy, with a sensitivity and specificity of 89.0% and 85.0%, whereas uptake less than 100 units appears to correlate with a benign pathology., Limitations: The limited sample size, the physiological uptake, and excretion of indocyanine green might interfere with the assessment of unnoticed implants in the bowel serosa and liver., Conclusions: Quantitative indocyanine green seems to be useful for the assessment of nonmucinous colorectal peritoneal metastases. Fluorescence uptake greater than 181 units appears to correlate with malignancy, whereas uptake less than 100 units appears to correlate with a benign pathology. See Video Abstract at http://links.lww.com/DCR/B743., Evaluacin Cuantitativa De Imgenes De Fluorescencia Con Verde De Indocianina Para Metstasis Peritoneales No Mucinosas Resultados Preliminares Del Estudio Iccp: ANTECEDENTES:En pacientes seleccionados con metástasis peritoneales de origen colorrectal, la citorreducción com-pleta ha sido el único factor pronóstico principal que influye en el resultado a largo plazo. En estos pacientes, las imágenes de fluorescencia con verde de indocianina parecen ser útiles para detectar pequeños implantes peritoneales subclínicos. Sin embargo, el análisis cuantitativo de fluorescencia aún no se ha establecido como estándar.OBJETIVO:Evaluar la sensibilidad y especificidad de la evaluación cuantitativa de fluorescencia verde de indo-cianina, en la detección de metástasis peritoneales de origen colorrectal no mucinoso.DISEÑO:Ensayo prospectivo de intervención baja de un solo brazo y un solo centro.ENTORNO CLINICO:El dispositivo se utilizó para la evaluación cuantitativa de fluorescencia intraoperatoria.PACIENTES:Pacientes consecutivos diagnosticados con metástasis peritoneales de origen colorrectal, selecciona-dos para cirugía curativa y que cumplieron con los criterios de inclusión.INTERVENCIONES:Se administró verde de indocianina por vía intravenosa 12 h antes de la cirugía. La citorreducción se realizó mediante identificación de nódulos con luz blanca y luego con verde de indocianina. Final-mente, se evaluó la fluorescencia ex vivo.PRINCIPALES MEDIDAS DE VALORACION:Sensibilidad y especificidad cuantitativa de la fluorescencia.RESULTADOS:Los primeros 11 pacientes fueron incluidos en este análisis preliminar. En total se resecaron 52 nódu-los, siendo 37 (71,1%) diagnosticados como malignos en el análisis histopatológico. De ellos, 5 (13,5%) eran indetectables bajo luz blanca y solamente se identificaron con fluorescencia. Se detec-taron un total de 15 nódulos no malignos bajo luz blanca, de los cuales 8 (53,3%) fueron fluorescen-tes negativos. La fluorescencia superior a 181 unidades podría ser el umbral de malignidad, con una sensibilidad y especificidad del 89,0% y el 85,0% respectivamente; mientras que la captación por debajo de 100 unidades parece correlacionarse con una patología benigna.LIMITACIONES:El tamaño limitado de la muestra; la captación fisiológica y la excreción de verde de indocianina pueden interferir con la evaluación de implantes inadvertidos en la serosa intestinal y el hígado.CONCLUSIONES:La cuantificación del verde de indocianina, parece ser útil en la evaluación de metástasis peritonea-les colorrectales no mucinosas. La captación de fluorescencia por encima de 181 unidades parece correlacionarse con la malignidad, mientras que la captación por debajo de 100 unidades parece co-rrelacionarse con una patología benigna. Consulte Video Resumen en http://links.lww.com/DCR/B743. (Traducción - Dr. Fidel Ruiz Healy)., Competing Interests: Financial Disclosure: Drs González-Abós, Besa, Borja de Lacy, Valverde, and Almenara have no conflicts of interest or financial ties to disclose. Dr Lacy reports personal fees from Medtronic, Olympus, Applied Medical, and Conmed outside of the submitted work., (Copyright © The ASCRS 2021.)

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, the step of exogenous staining makes these methods undesirable for rapid, nondestructive, and long-term investigation. Here, we present an instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity. This concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and nucleus area for the labeled and unlabeled populations reveal that the chemical reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals to assessing the effectiveness of cancer treatments., (© 2022. The Author(s).)

Competing Interests: Competing interests: None declared.

A foveated display is a technology that can solve the problem of insufficient angular resolution (relative to the human eye) for near-eye display. In a high-resolution foveated display, a beam steering element is required to track the human gaze. An electrowetting prism array is a transmissive non-mechanical beam steering device, that allows a light and compact optical system to be configured and a large aperture possible. However, the view is obstructed by the sidewall of the prism array. When the size of the cell prism is 7mm, the prism array has an 87% fill-factor. To push the fill-factor to 100%, the cell prisms were magnified using a lens array. Image processing was performed such that the image produced by the lens array was identical to the original. Beam steering by refraction is accompanied by chromatic dispersion, which causes chromatic aberration, making colors appear blurry. The refractive index condition to reduce chromatic dispersion was obtained using the doublet structure of the electrowetting prism. The chromatic dispersion was reduced by 70% on average.

Foveated imaging, which has the ability to provide overall situational awareness over a large field of view and high-resolution perception of local details, has significant advantages in many specific applications. However, existing artificially foveated imaging systems are complex, bulky, and expensive, and the flexibility of the fovea specifically has many limitations. To overcome these deficiencies, this paper proposes a method for foveated imaging by collecting multiple partially overlapping sub-fields of view. To capture the above special sub-fields of view, we propose a high-efficiency algorithm based on the characteristics of the field of view deflected by the Risley-prism and aimed at solving the prism rotation angles. In addition, we prove the reliability of the proposed algorithm by cross-validation with the particle swarm optimization algorithm. The experimental results show that the proposed method can achieve flexible foveated imaging using a single Risley-prism imaging system.

Waveguide-type near-eye displays have useful properties such as compact form factor, lightweight and see-through capability. Conventional systems, however, support only a single image plane fixed at a certain distance, which may induce eye fatigue due to the vergence-accommodation conflict. In this paper, we propose a waveguide-type near-eye display with two image planes using a polarization grating. Two images with orthogonal polarizations propagate within the waveguide with different total internal reflection angles and form virtual images at different distances. The use of the polarization grating and two pairs of holographic optical elements enables dual image plane formation by a single waveguide with high transparency for the real scene. Optical experiments confirm the principle of the proposed optical system.

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm 2 , encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain., (© 2021. The Author(s).)

Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales., (© 2021. The Author(s).)

The use of optical techniques to interrogate wide ranging samples from semiconductors to biological tissue for rapid analysis and diagnostics has gained wide adoption over the past decades. The desire to collect ever more spatially, spectrally and temporally detailed optical signatures for sample characterization has specifically driven a sharp rise in new optical microscopy technologies. Here we present a high-speed optical scanning microscope capable of capturing time resolved images across 512 spectral and 32 time channels in a single acquisition with the potential for ~0.2 frames per second (256 × 256 image pixels). Each pixel in the resulting images contains a detailed data cube for the study of diverse time resolved light driven phenomena. This is enabled by integration of system control electronics and on-chip processing which overcomes the challenges presented by high data volume and low imaging speed, often bottlenecks in previous systems., (© 2021. The Author(s).)

High positive margin rates in oncologic breast-conserving surgery are a pressing clinical problem. Volumetric X-ray scanning is emerging as a powerful ex vivo specimen imaging technique for analyzing resection margins, but X-rays lack contrast between non-malignant and malignant fibrous tissues. In this study, combined micro-CT and wide-field optical image radiomics were developed to classify malignancy of breast cancer tissues, demonstrating that X-ray/optical radiomics improve malignancy classification. Ninety-two standardized features were extracted from co-registered micro-CT and optical spatial frequency domain imaging samples extracted from 54 breast tumors exhibiting seven tissue subtypes confirmed by microscopic histological analysis. Multimodal feature sets improved classification performance versus micro-CT alone when adipose samples were included (AUC = 0.88 vs. 0.90; p-value = 3.65e-11) and excluded, focusing the classification task on exclusively non-malignant fibrous versus malignant tissues (AUC = 0.78 vs. 0.85; p-value = 9.33e-14). Extending the radiomics approach to high-dimensional optical data-termed "optomics" in this study-offers a promising optical image analysis technique for cancer detection. Radiomic feature data and classification source code are publicly available., (© 2021. The Author(s).)

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells' lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution., (© 2021. The Author(s).)

Infrared upconversion devices (UCDs) enable low-cost visualization of infrared optical signals without utilizing a readout circuit, which is of great significance for biological recognition and noninvasive dynamic monitoring. However, UCDs suffer from inferior photon to photon (p-p) efficiency and high turn-on voltage (V on ) for upconversion operation, hindering a further expansion in highly resolved infrared imaging. Herein, an efficient organic UCD integrating an interfacial exciplex emitter and a well-designed near-infrared (NIR) detector reveals a high efficiency up to 12.92% and a low V on down to 1.56 V. The low V on gives the capacity for detecting weak NIR light down to 3.2 µW cm -2 , significantly expanding the detection power scale of UCDs. Thus, the imaging linear dynamic range (I-LDR) is highly bias-tunable, ranging from 13.23 to 84.4 dB. The high I-LDR enables highly resolved and strong-penetration bioimaging especially for thick biological sections, indicating great potential in noninvasive defect and pathological detection., (© 2021 Wiley-VCH GmbH.)

Over the past years, broadband achromatic metalenses have been intensively studied due to their great potential for applications in consumer and industry products. Even though significant progress has been made, the efficiency of technologically relevant silicon metalenses is limited by the intrinsic material loss above the bandgap. In turn, the recently proposed achromatic metalens utilizing transparent, high-index materials such as titanium dioxide has been restricted by the small thickness and showed relatively low focusing efficiency at longer wavelengths. Consequently, metalens-based optical imaging in the biological transparency window has so far been severely limited. Herein, we experimentally demonstrate a polarization-insensitive, broadband titanium dioxide achromatic metalens for applications in the near-infrared biological imaging. A large-scale fabrication technology has been developed to produce titanium dioxide nanopillars with record-high aspect ratios featuring pillar heights of 1.5 µm and ~90° vertical sidewalls. The demonstrated metalens exhibits dramatically increased group delay range, and the spectral range of achromatism is substantially extended to the wavelength range of 650-1000 nm with an average efficiency of 77.1%-88.5% and a numerical aperture of 0.24-0.1. This research paves a solid step towards practical applications of flat photonics., (© 2021. The Author(s).)

Laser scanning is used in advanced biological microscopy to deliver superior imaging contrast, resolution and sensitivity. However, it is challenging to scale up the scanning speed required for interrogating a large and heterogeneous population of biological specimens or capturing highly dynamic biological processes at high spatiotemporal resolution. Bypassing the speed limitation of traditional mechanical methods, free-space angular-chirp-enhanced delay (FACED) is an all-optical, passive and reconfigurable laser-scanning approach that has been successfully applied in different microscopy modalities at an ultrafast line-scan rate of 1-80 MHz. Optimal FACED imaging performance requires optimized experimental design and implementation to enable specific high-speed applications. In this protocol, we aim to disseminate information allowing FACED to be applied to a broader range of imaging modalities. We provide (i) a comprehensive guide and design specifications for the FACED hardware; (ii) step-by-step optical implementations of the FACED module including the key custom components; and (iii) the overall image acquisition and reconstruction pipeline. We illustrate two practical imaging configurations: multimodal FACED imaging flow cytometry (bright-field, fluorescence and second-harmonic generation) and kHz 2D two-photon fluorescence microscopy. Users with basic experience in optical microscope operation and software engineering should be able to complete the setup of the FACED imaging hardware and software in ~2-3 months., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Acousto-optic (AO) imaging is an in-depth optical imaging technique of highly scattering media. One challenging end-application for this technique is to perform imaging of living biological tissues. Indeed, because it relies on coherent illumination, AO imaging is sensitive to speckle decorrelation occurring on the millisecond time scale. Camera-based detections are well suited for in vivo imaging provided their integration time is lower than those decorrelation time scales. We present Fourier transform acousto-optic imaging combined with off-axis holography, which relies on plane waves and long-duration pulses. We demonstrate, for the first time to the best of our knowledge, a two-dimensional imaging system fully compatible with in vivo imaging prerequisites. The method is validated experimentally by performing in-depth imaging inside a multiple scattering sample.

We propose a new, to the best of our knowledge, compound technique to measure high-dynamic-range blood flow rate in a large-diameter vessel, which combines the dynamic scattering light (DLS) and the laser speckle contrast imaging (LSCI) methods, possessing the advantages of the high temporal resolution of DLS and the robust property of LSCI. By controlling the second-order spatial correlations of the laser speckle through two imaging systems, the speckle temporal intensity autocorrelation function g 2 ( t ) and the decorrelation time τ c are directly measured using a high-speed camera. It turns out the enhanced spatial second-order correlation helps to measure the blood flow with higher dynamic range and that the measured parameter β and the blood flow dynamics n were accurately determined. For further improvement the dynamic range, the modified LSCI method was adopted, and the decorrelation time as a function of blood flow rate was constructed. It reveals the feasibility of measuring the high flow rate in large-diameter vessels and provides significant guidance for the future biomedical study of the myocardial perfusion in coronary artery bypass grafting, ghost imaging, and ghost cytometry.

Single-pixel holography (SPH) is capable of generating holographic images with rich spatial information by employing only a single-pixel detector. Thanks to the relatively low dark-noise production, high sensitivity, large bandwidth, and cheap price of single-pixel detectors in comparison to pixel-array detectors, SPH is becoming an attractive imaging modality at wavelengths where pixel-array detectors are not available or prohibitively expensive. In this work, we develop a high-throughput single-pixel compressive holography with a space-bandwidth-time product (SBP-T) of 41,667 pixels/s, realized by enabling phase stepping naturally in time and abandoning the need for phase-encoded illumination. This holographic system is scalable to provide either a large field of view (~83 mm 2 ) or a high resolution (5.80 μm × 4.31 μm). In particular, high-resolution holographic images of biological tissues are presented, exhibiting rich contrast in both amplitude and phase. This work is an important step towards multi-spectrum imaging using a single-pixel detector in biophotonics., (© 2021. The Author(s).)

Topological characterization of the Retinal microvascular nEtwork visualized by portable fuNDus camera (TREND) is a database comprising of 72 color digital retinal images collected from the students of the Faculty of Medicine at the University of Montenegro, in the period from February 18th to March 11th 2020. The database also includes binarized images of manually segmented microvascular networks associated with each raw image. The participant demographic characteristics, health status, and social habits information such as age, sex, body mass index, smoking history, alcohol use, as well as previous medical history was collected. As proof of the concept, a smaller set of 10 color digital fundus images from healthy older participants is also included. Comparison of the microvascular parameters of these two sets of images demonstrate that digital fundus images recorded with a hand-held portable camera are able to capture the changes in patterns of microvascular network associated with aging. The raw images from the TREND database provide a standard that defines normal retinal anatomy and microvascular network geometry in young healthy people in Montenegro as it is seen with the digital hand-held portable non-mydriatic MiiS HORUS Scope DEC 200.This knowledge could facilitate the application of this technology at the primary level of health care for large scale telematic screening for complications of chronic diseases, such as hypertensive and diabetic retinopathy. In addition, it could aid in the development of new methods for early detection of age-related changes in the retina, systemic chronic diseases, as well as eye-specific diseases. The associated manually segmented images of the microvascular networks provide the standard that can be used for development of automatic software for image quality assessment, segmentation of microvascular network, and for computer-aided detection of pathological changes in retina. The TREND database is freely available at https://doi.org/10.5281/zenodo.4521043., Competing Interests: The authors have declared that no competing interests exist.

Background: Re-excision due to positive margins following breast-conserving surgery (BCS) negatively affects patient outcomes and healthcare costs. The inability to visualize margin involvement is a significant challenge in BCS. 5-Aminolevulinic acid hydrochloride (5-ALA HCl), a non-fluorescent oral prodrug, causes intracellular accumulation of fluorescent porphyrins in cancer cells. This single-center Phase II randomized controlled trial evaluated the safety, feasibility, and diagnostic accuracy of a prototype handheld fluorescence imaging device plus 5-ALA for intraoperative visualization of invasive breast carcinomas during BCS., Methods: Fifty-four patients were enrolled and randomized to receive no 5-ALA or oral 5-ALA HCl (15 or 30 mg/kg). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed, and biopsies were collected from within and outside the clinically demarcated tumor border of the gross specimen for blinded histopathology., Results: In the absence of 5-ALA, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Both 5-ALA doses caused bright red tumor fluorescence, with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA group, the positive predictive value (PPV) for detecting breast cancer inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively. In the 30 mg/kg 5-ALA group, the PPV was 100.0% and 50.0% inside and outside the demarcated tumor border, respectively. No adverse events were observed, and clinical feasibility of this imaging device-5-ALA combination approach was confirmed., Conclusions: This is the first known clinical report of visualization of 5-ALA-induced fluorescence in invasive breast carcinoma using a real-time handheld intraoperative fluorescence imaging device., Trial Registration: Clinicaltrials.gov identifier NCT01837225 . Registered 23 April 2013., (© 2021. The Author(s).)

Background: Gastric inflammation is a major risk factor for gastric cancer. Current endoscopic methods are not able to efficiently detect and characterize gastric inflammation, leading to a sub-optimal patients' care. New non-invasive methods are needed. Reflectance mucosal light analysis is of particular interest in this context. The aim of our study was to analyze reflectance light and specific autofluorescence signals, both in humans and in a mouse model of gastritis., Methods: We recruited patients undergoing gastroendoscopic procedure during which reflectance was analysed with a multispectral camera. In parallel, the gastritis mouse model of Helicobacter pylori infection was used to investigate reflectance from ex vivo gastric samples using a spectrometer. In both cases, autofluorescence signals were measured using a confocal microscope., Findings: In gastritis patients, reflectance modifications were significant in near-infrared spectrum, with a decrease between 610 and 725 nm and an increase between 750 and 840 nm. Autofluorescence was also modified, showing variations around 550 nm of emission. In H. pylori infected mice developing gastric inflammatory lesions, we observed significant reflectance modifications 18 months after infection, with increased intensity between 617 and 672 nm. Autofluorescence was significantly modified after 1, 3 and 6 months around 550 and 630 nm. Both in human and in mouse, these reflectance data can be considered as biomarkers and accurately predicted inflammatory state., Interpretation: In this pilot study, using a practical measuring device, we identified in humans, modification of reflectance spectra in the visible spectrum and for the first time in near-infrared, associated with inflammatory gastric states. Furthermore, both in the mouse model and humans, we also observed modifications of autofluorescence associated with gastric inflammation. These innovative data pave the way to deeper validation studies on larger cohorts, for further development of an optical biopsy system to detect gastritis and finally to better surveil this important gastric cancer risk factor., Funding: The project was funded by the ANR EMMIE (ANR-15-CE17-0015) and the French Gastroenterology Society (SNFGE)., Competing Interests: Declaration of Competing Interest Authors have no conflicts of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

In this paper, we present the imaging formation process of the piecewise mirror eyes of the deep-sea spookfish, which has a strange combination of refractive and reflective eyes. The biological reflective eye structure is formulated to the curved surface's flat mirror array. Zemax is utilized to evaluate optical features such as the modulation transfer function, distortion, and imaging performances. However, the natural images are highly distorted, and the resolution is lower than expected. Therefore, we increase the number of piecewise mirrors of the fisheye to see higher quality images, which can be improved entirely by the mirror shapes. Finally, the fisheye's imaging analysis reveals the deep-sea creature's resolution limit and also shows the possibility of artificial and biomimetic camera applications.