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Significance: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue. Aim: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing. Approach: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications. Results: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality. Conclusions: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

Significance: Tissue simulating phantoms are an important part of validating biomedical optical techniques. Tissue pathology in inflammation and oedema involves changes in both water and hemoglobin fractions. Aim: We present a method to create solid gelatin-based phantoms mimicking inflammation and oedema with adjustable water and hemoglobin fractions. Approach: One store-bought gelatin and one research grade gelatin were evaluated. Different water fractions were obtained by varying the water-to-gelatin ratio. Ferrous stabilized human hemoglobin or whole human blood was added as absorbers, and the stability and characteristics of each were compared. Intralipid® was used as the scatterer. All phantoms were characterized using spatial frequency domain spectroscopy. Results: The estimated water fraction varied linearly with expected values (R2 = 0.96 for the store-bought gelatin and R2 = 0.99 for the research grade gelatin). Phantoms including ferrous stabilized hemoglobin stayed stable up to one day but had methemoglobin present at day 0. The phantoms with whole blood remained stable up to 3 days using the store-bought gelatin. Conclusions: A range of physiological relevant water fractions was obtained for both gelatin types, with the stability of the phantoms including hemoglobin differing between the gelatin type and hemoglobin preparation. These low-cost phantoms can incorporate other water-based chromophores and be fabricated as thin sheets to form multilayered structures. Funding: This research was financially supported by Knut and Alice Wallenberg Foundation’s Center for Molecular Medicine at Linkoping University (WCMM) and Hudfonden’s Edvard Welander och Finsenstiftelsen.

Significance: Intravascular photoacoustic (IVPA) imaging can identify native lipid in atherosclerotic plaques in vivo. However, the large number of laser pulses required to produce 3D images is a safety concern that has not been fully addressed. Aim: We aim to evaluate if irradiation at wavelengths and dosages relevant to IVPA imaging causes target vessel damage. Approach: We irradiate the carotid artery of swine at one of several energy dosages using radiation at 1064 or 1720 nm and use histological evaluation by a pathologist to identify dose-dependent damage. Results: Media necrosis was the only dose-dependent form of injury. Damage was present at a cumulative fluence of 50 J/cm2 when using 1720 nm light. Damage was more equivocally identified at 700 J/cm2 using 1064 nm. Conclusions: In prior work, IVPA imaging of native lipid in swine has been successfully conducted below the damage thresholds identified. This indicates that it will be possible to use IVPA imaging in a clinical setting without damaging vessel tissue. Future work should determine if irradiation causes an increase in blood thrombogenicity and confirm whether damaged tissue will heal over longer time points.

Significance: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging. Aim: To quantify and classify complex collagen fiber organizations. Approach: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy U, enstrophy E, and tortuosity tau. Results: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures. Conclusions: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for E and U increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of tau cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Published version

Searching analyzable metaphase chromosomes is a critical step for the diagnosis and treatment of leukemia patients, and the searching efficiency is limited by the difficulty that the conventional microscopic systems have in simultaneously achieving high resolution and a large field of view (FOV). However, this challenge can be addressed by Fourier ptychography microscopy (FPM) technology.The purpose of this study is to investigate the feasibility of utilizing FPM to reconstruct high-resolution chromosome images.An experimental FPM prototype, which was equipped with 4 × / 0.1 NA or 10 × / 0.25 NA objective lenses to achieve a theoretical equivalent NA of 0.48 and 0.63, respectively, was developed. Under these configurations, we first generated the system modulation transfer function (MTF) curves to assess the resolving power. Next, a group of analyzable metaphase chromosomes were imaged by the FPM system, which were acquired from the peripheral blood samples of the leukemia patients. The chromosome feature qualities were evaluated and compared with the results accomplished by the corresponding conventional microscopes.The MTF curve results indicate that the resolving power of the 4 × / 0.1 NA FPM system is equivalent and comparable to the 20 × / 0.4 NA conventional microscope, whereas the performance of the 10 × / 0.25 NA FPM system is close to the 60 × / 0.95 NA conventional microscope. When imaging the chromosomes, the feature qualities of the 4 × / 0.1 NA FPM system are comparable to the results under the conventional 20 × / 0.4 NA lens, whereas the feature qualities of the 10 × / 0.25 NA FPM system are better than the conventional 60 × / 0.95 NA lens and comparable to the conventional 100 × / 1.25 NA lens.This study initially verified that it is feasible to utilize FPM to develop a high-resolution and wide-field chromosome sample scanner.

Significance: Optical properties (absorption coefficient and scattering coefficient) of tissue are the most critical parameters for disease diagnosis-based optical method. In recent years, researchers proposed spatial frequency domain imaging (SFDI) to quantitatively map tissue optical properties in a broad field of contactless imaging. To solve the limitations in wavebands unsuitable for silicon-based sensor technology, a compressed sensing (CS) algorithm is used to reproduce the original signal by a single-pixel detectors. Currently, the existing single-pixel SFDI method mainly uses a random sampling policy to extract and recover signals in the acquisition stage. However, these methods are memory-hungry and time-consuming, and they cannot generate discernible results under low sampling rate. Explorations on high performance and efficiency single-pixel SFDI are of great significance for clinical application. Aim: Fourier single-pixel imaging can reconstruct signals with less time and space costs and has fewer reconstruction errors. We focus on an SFDI algorithm based on Fourier single-pixel imaging and propose our Fourier single-pixel image-based spatial frequency domain imaging method (FSI-SFDI). Approach: First, we use Fourier single-pixel imaging algorithm to collect and compress signals and SFDI algorithm to generate optical parameters. Given the basis that the main energy of general image signals is concentrated in the range of low frequency of Fourier frequency domain, our FSI-SFDI uses a circular-sampling scheme to sample data points in the low-frequency region. Then, we reconstruct the image details from these points by optimization-based inverse-FFT method. Results: Our algorithm is tested on simulated data. Results show that the root mean square error (RMSE) of optical parameters is lower than 5% when the data reduction is 92%, and it can generate discernible optical parameter image with low sampling rate. We can observe that our FSI-SFDI primarily recovers the optical properties while keeping the RMSE under the upper bound of 4.5% when we use an image with 512×512 resolution as the example for calculation and analysis. Not only that but also our algorithm consumes less space and time for an image with 256×256 resolution, the signal reconstruction takes only 1.65 ms, and requires less RAM memory. Compared to CS-SFDI method, our FSI-SFDI can reduce the required number of measurements through optimizing algorithm. Conclusions: Moreover, FSI-SFDI is capable of recovering high-quality resolvable images with lower sampling rate, higher-resolution images with less memory and time consumed than previous CS-SFDI method, which is very promising for clinical data collection and medical analysis.

Significance: An effective contrast agent for concurrent multimodal photoacoustic (PA) and ultrasound (US) imaging must have both high optical absorption and high echogenicity. Integrating a highly absorbing dye into the lipid shell of gas core nanobubbles (NBs) adds PA contrast to existing US contrast agents but may impact agent ultrasonic response. Aim: We report on the development and ultrasonic characterization of lipid-shell stabilized C3F8 NBs with integrated Sudan Black (SB) B dye in the shell as dual-modal PA-US contrast agents. Approach: Perfluoropropane NBs stabilized with a lipid shell including increasing concentrations of SB B dye were formulated by amalgamation (SBNBs). Physical properties of SBNBs were characterized using resonant mass measurement, transmission electron microscopy and pendant drop tensiometry. Concentrated bubble solutions were imaged for 8 min to assess signal decay. Diluted bubble solutions were stimulated by a focused transducer to determine the response of individual NBs to long cycle (30 cycle) US. For assessment of simultaneous multimodal contrast, bulk populations of SBNBs were imaged using a PA and US imaging platform. Results: We produced high agent yield (∼1011) with a mean diameter of ∼200 to 300 nm depending on SB loading. A 40% decrease in bubble yield was measured for solutions with 0.3 and 0.4 mg/ml SB. The addition of SB to the shell did not substantially affect NB size despite an increase in surface tension by up to 8 mN/m. The bubble decay rate increased after prolonged exposure (8 min) by dyed bubbles in comparison to their undyed counterparts (2.5-fold). SB in bubble shells increased gas exchange across the shell for long cycle US. PA imaging of these agents showed an increase in power (up to 10 dB) with increasing dye. Conclusions: We added PA contrast function to NBs. The addition of SB increased gas exchange across the NB shell. This has important implications in their use as multimodal agents.

Significance: Tissue birefringence is an important parameter to consider when designing realistic, tissue-mimicking phantoms. Options for suitable birefringent materials that can be used to accurately represent tissue scattering are limited. Aim: To introduce a method of fabricating birefringent tissue phantoms with a commonly used material—polydimethylsiloxane (PDMS)—for imaging with polarization-sensitive optical coherence tomography (PS-OCT). Approach: Stretch-induced birefringence was characterized in PDMS phantoms made with varying curing ratios, and the resulting phantom birefringence values were compared with those of biological tissues. Results: We showed that, with induced birefringence levels up to 2.1×10−4, PDMS can be used to resemble the birefringence levels in weakly birefringent tissues. We demonstrated the use of PDMS in the development of phantoms to mimic the normal and diseased bladder wall layers, which can be differentiated by their birefringence levels. Conclusions: PDMS allows accurate control of tissue scattering and thickness, and it exhibits controllable birefringent properties. The use of PDMS as a birefringent phantom material can be extended to other birefringence imaging systems beyond PS-OCT and to mimic other organs.

Significance: In order to elucidate therapeutic treatment to accelerate wound healing, it is crucial to understand the process underlying skin wound healing, especially re-epithelialization. Epidermis and scab detection is of importance in the wound healing process as their thickness is a vital indicator to judge whether the re-epithelialization process is normal or not. Since optical coherence tomography (OCT) is a real-time and non-invasive imaging technique that can perform a cross-sectional evaluation of tissue microstructure, it is an ideal imaging modality to monitor the thickness change of epidermal and scab tissues during wound healing processes in micron-level resolution. Traditional segmentation on epidermal and scab regions was performed manually, which is time-consuming and impractical in real time. Aim: We aim to develop a deep-learning-based skin layer segmentation method for automated quantitative assessment of the thickness of in vivo epidermis and scab tissues during a time course of healing within a rodent model. Approach: Five convolution neural networks were trained using manually labeled epidermis and scab regions segmentation from 1000 OCT B-scan images (assisted by its corresponding angiographic information). The segmentation performance of five segmentation architectures was compared qualitatively and quantitatively for validation set. Results: Our results show higher accuracy and higher speed of the calculated thickness compared with human experts. The U-Net architecture represents a better performance than other deep neural network architectures with 0.894 at F1-score, 0.875 at mean intersection over union, 0.933 at Dice similarity coefficient, and 18.28 μm at an average symmetric surface distance. Furthermore, our algorithm is able to provide abundant quantitative parameters of the wound based on its corresponding thickness maps in different healing phases. Among them, normalized epidermal thickness is recommended as an essential hallmark to describe the re-epithelialization process of the rodent model. Conclusions: The automatic segmentation and thickness measurements within different phases of wound healing data demonstrates that our pipeline provides a robust, quantitative, and accurate method for serving as a standard model for further research into effect of external pharmacological and physical factors.

Significance: Time-domain functional near-infrared spectroscopy (TD-fNIRS) has been considered as the gold standard of noninvasive optical brain imaging devices. However, due to the high cost, complexity, and large form factor, it has not been as widely adopted as continuous wave NIRS systems. Aim: Kernel Flow is a TD-fNIRS system that has been designed to break through these limitations by maintaining the performance of a research grade TD-fNIRS system while integrating all of the components into a small modular device. Approach: The Kernel Flow modules are built around miniaturized laser drivers, custom integrated circuits, and specialized detectors. The modules can be assembled into a system with dense channel coverage over the entire head. Results: We show performance similar to benchtop systems with our miniaturized device as characterized by standardized tissue and optical phantom protocols for TD-fNIRS and human neuroscience results. Conclusions: The miniaturized design of the Kernel Flow system allows for broader applications of TD-fNIRS.

Significance: Despite the ample progress made toward faster and more accurate Monte Carlo (MC) simulation tools over the past decade, the limited usability and accessibility of these advanced modeling tools remain key barriers to widespread use among the broad user community. Aim: An open-source, high-performance, web-based MC simulator that builds upon modern cloud computing architectures is highly desirable to deliver state-of-the-art MC simulations and hardware acceleration to general users without the need for special hardware installation and optimization. Approach: We have developed a configuration-free, in-browser 3D MC simulation platform—Monte Carlo eXtreme (MCX) Cloud—built upon an array of robust and modern technologies, including a Docker Swarm-based cloud-computing backend and a web-based graphical user interface (GUI) that supports in-browser 3D visualization, asynchronous data communication, and automatic data validation via JavaScript Object Notation (JSON) schemas. Results: The front-end of the MCX Cloud platform offers an intuitive simulation design, fast 3D data rendering, and convenient simulation sharing. The Docker Swarm container orchestration backend is highly scalable and can support high-demand GPU MC simulations using MCX over a dynamically expandable virtual cluster. Conclusion: MCX Cloud makes fast, scalable, and feature-rich MC simulations readily available to all biophotonics researchers without overhead. It is fully open-source and can be freely accessed at http://mcx.space/cloud.

Significance: Convolutional neural networks (CNNs) show the potential for automated classification of different cancer lesions. However, their lack of interpretability and explainability makes CNNs less than understandable. Furthermore, CNNs may incorrectly concentrate on other areas surrounding the salient object, rather than the network’s attention focusing directly on the object to be recognized, as the network has no incentive to focus solely on the correct subjects to be detected. This inhibits the reliability of CNNs, especially for biomedical applications. Aim: Develop a deep learning training approach that could provide understandability to its predictions and directly guide the network to concentrate its attention and accurately delineate cancerous regions of the image. Approach: We utilized Selvaraju et al.’s gradient-weighted class activation mapping to inject interpretability and explainability into CNNs. We adopted a two-stage training process with data augmentation techniques and Li et al.’s guided attention inference network (GAIN) to train images captured using our customized mobile oral screening devices. The GAIN architecture consists of three streams of network training: classification stream, attention mining stream, and bounding box stream. By adopting the GAIN training architecture, we jointly optimized the classification and segmentation accuracy of our CNN by treating these attention maps as reliable priors to develop attention maps with more complete and accurate segmentation. Results: The network’s attention map will help us to actively understand what the network is focusing on and looking at during its decision-making process. The results also show that the proposed method could guide the trained neural network to highlight and focus its attention on the correct lesion areas in the images when making a decision, rather than focusing its attention on relevant yet incorrect regions. Conclusions: We demonstrate the effectiveness of our approach for more interpretable and reliable oral potentially malignant lesion and malignant lesion classification.

Significance: Current methods for analyzing pathological muscle tissue are time consuming and rarely quantitative, and they involve invasive biopsies. Faster and less invasive diagnosis of muscle disease may be achievable using marker-free in vivo optical sensing methods. Aim: It was speculated that changes in the biochemical composition and structure of muscle associated with pathology could be measured quantitatively using visible wavelength optical spectroscopy techniques enabling automated classification. Approach: A fiber-optic autofluorescence (AF) and diffuse reflectance (DR) spectroscopy device was manufactured. The device and data processing techniques based on principal component analysis were validated using in situ measurements on healthy skeletal and cardiac muscle. These methods were then applied to two mouse models of genetic muscle disease: a type 1 neurofibromatosis (NF1) limb-mesenchyme knockout (Nf1Prx1−/−) and a muscular dystrophy mouse (mdx). Results: Healthy skeletal and cardiac muscle specimens were separable using AF and DR with receiver operator curve areas (ROC-AUC) of >0.79. AF and DR analyses showed optically separable changes in Nf1Prx1−/− quadriceps muscle (ROC-AUC >0.97) with no differences detected in the heart (ROC-AUC

Significance: Light-field fundus photography has the potential to be a new milestone in ophthalmology. Up-to-date publications show only unsatisfactory image quality, preventing the use of depth measurements. We show that good image quality and, consequently, reliable depth measurements are possible, and we investigate the current challenges of this novel technology. Aim: We investigated whether light field (LF) imaging of the retina provides depth information, on which structures the depth is estimated, which illumination wavelength should be used, whether deeper layers are measurable, and what kinds of artifacts occur. Approach: The technical setup, a mydriatic fundus camera with an LF imager, and depth estimation were validated by an eye model and in vivo measurements of three healthy subjects and three subjects with suspected glaucoma. Comparisons between subjects and the corresponding optical coherence tomography (OCT) measurements were used for verification of the depth estimation. Results: This LF setup allowed for three-dimensional one-shot imaging and depth estimation of the optic disc with green light. In addition, a linear relationship was found between the depth estimates of the OCT and those of the setup developed here. This result is supported by the eye model study. Deeper layers were not measurable. Conclusions: If image artifacts can be handled, LF technology has the potential to help diagnose and monitor glaucoma risk at an early stage through a rapid, cost-effective one-shot technology.

Significance: For optical methods to accurately assess hemoglobin oxygen saturation in vivo, an independently verifiable tissue-like standard is required for validation. For this purpose, we propose three hemoglobin preparations and evaluate methods to characterize them. Aim: To spectrally characterize three different hemoglobin preparations using multiple spectroscopic methods and to compare their absorption spectra to commonly used reference spectra. Approach: Absorption spectra of three hemoglobin preparations in solution were characterized using spectroscopic collimated transmission: whole blood, lysed blood, and ferrous-stabilized hemoglobin. Tissue-mimicking phantoms composed of Intralipid, and the hemoglobin solutions were characterized using spatial frequency-domain spectroscopy (SFDS) and enhanced perfusion and oxygen saturation (EPOS) techniques while using yeast to deplete oxygen. Results: All hemoglobin preparations exhibited similar absorption spectra when accounting for methemoglobin and scattering in their oxyhemoglobin and deoxyhemoglobin forms, respectively. However, systematic differences were observed in the fitting depending on the reference spectra used. For the tissue-mimicking phantoms, SFDS measurements at the surface of the phantom were affected by oxygen diffusion at the interface with air, associated with higher values than for the EPOS system. Conclusions: We show the validity of different blood phantoms and what considerations need to be addressed in each case to utilize them equivalently. Funding: This research was financially supported by VINNOVA grants via the Swelife and MedTech4Health programs (Grant Nos. 2016-02211, 2017-01435, and 2019-01522) and the Knut and Alice Wallenberg Foundation’s Center for Molecular Medicine at Linköping University (WCMM).

Significance: Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans. Aim: We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes. Approach: A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet. Results: On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and 2.4 μm in the lateral and axial directions, respectively. Conclusion: The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

Significance: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies. Aim: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces. Approach: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise. Results: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint. Conclusions: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.

Significance: Numerous optical imaging and spectroscopy techniques are used to study the tissue-optical properties; the majority of them are limited in information regarding the penetration depth. A simple, safe, easily applicable diagnostic technique is required to get deeper tissue information in a multilayer structure. Aim: A fiber-based diffuse reflectance (DR) technique is used to extract and quantify the bottom layer absorption coefficients in two-layer (2L) tissue-mimicking solid phantoms. We determine the Indian black ink concentrations in a deep-hidden layer that is sandwiched between agar and silicone-based phantom layers. Approach: A fiber-based DR experiment was performed to study the optical properties of the tissue at higher penetration depth, with different fiber core diameters and a constant numerical aperture (0.5 NA). The optimal core diameter of the fiber was chosen by measuring solid phantoms. In 2L phantoms, the thickness of the top layer was kept 5.5 mm with a constant absorption and reduced scattering coefficients (μa = 0.045 mm − 1 and μs ′ = 2.622 mm − 1), whereas the absorption coefficients of the bottom layers were varied from 0.014 to 0.037 mm − 1 keeping the μs ′ the same as the top layer. A unique crossover point (Cp) was found in the DR intensity profile against distance. We examined the slope before and after the Cp. These two slopes indicate the difference between the optical properties of the top and bottom layers. Our technique got further verification, as we successfully determined the Cp with different Indian black ink concentrations, placed at the junction between the agar and silicone-based phantom layers. Results: The DR measurements were applied to 2L phantoms. Two different slopes were found in 2L phantoms compared to the one-layer (optical properties equal to the top layer of 2L). We extracted the slopes before and after the Cp in the 2L phantoms. The calculated absorption coefficients before the Cp were 0.014 ± 0.0004, 0.022 ± 0.0003, 0.028 ± 0.0003, and 0.036 ± 0.0014 mm − 1, and the absorption coefficients after the Cp were 0.019 ± 0.0013, 0.013 ± 0.0004, 0.014 ± 0.0006, and 0.031 ± 0.0001 mm − 1, respectively. The calculated absorption coefficients before the Cp were in good agreement with the optical properties of the bottom layer. The calculated absorption coefficients after the Cp were not the same as the top layer. Our DR system successfully determines the crossover points 12.14 ± 0.11 and 11.73 ± 0.15 mm for 70% and 100% ink concentrations placed at the junction of the agar and silicone layers. Conclusions: In a 2L tissue structure, the Cp depends on the absorption coefficients of top and bottom layers and the thickness of the top layer. With the help of the Cp and the absorption coefficients, one can determine the thickness of the top layer or vice versa. The slope value before the Cp in the DR profile allowed us to determine the absorption properties of the bottom layer instead of having the average behavior of the 2L phantom in the far detection range (11.0 to 17.0 mm).

Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition. Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM. Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM. Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz. Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Significance: Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity. Aim: We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity. Approach: We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion. Results: We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy. Conclusions: Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.

Significance: High-speed three-dimensional (3D) super-resolution microscopy is a unique tool to investigate various biological phenomena; yet the technology is not broadly adopted due to its high cost and complex system design. Aim: We present a compact, low-cost, and high-speed 3D structured illumination microscopy (SIM) based on a digital micromirror device and binary holography to visualize fast biological events with super-resolution. Approach: The 3D SIM uses a digital micromirror device to generate three laser foci with individually controllable positions, phases, and amplitudes via binary holography at the back aperture of objective lens to form optimal 3D structured patterns. Fifteen raw images are sequentially recorded and processed by the 3D SIM algorithm to reconstruct a super-resolved image. Results: Super-resolution 3D imaging at a speed of 26.7 frames per second is achieved with a lateral and axial resolution of 155 and 487 nm, which corresponds to a 1.65- and 1.63-times resolution enhancement, respectively, comparing with standard deconvolution microscopy. Conclusions: The 3D SIM realizes fast super-resolution imaging with optimal 3D structured illumination, which may find important applications in biophotonics.

Significance: Peripheral pitting edema is a clinician-administered measure for grading edema. Peripheral edema is graded 0, 1 + , 2 + , 3 + , or 4 + , but subjectivity is a major limitation of this technique. A pilot clinical study for short-wave infrared (SWIR) molecular chemical imaging (MCI) effectiveness as an objective, non-contact quantitative peripheral edema measure is underway. Aim: We explore if SWIR MCI can differentiate populations with and without peripheral edema. Further, we evaluate the technology for correctly stratifying subjects with peripheral edema. Approach: SWIR MCI of shins from healthy subjects and heart failure (HF) patients was performed. Partial least squares discriminant analysis (PLS-DA) was used to discriminate the two populations. PLS regression (PLSR) was applied to assess the ability of MCI to grade edema. Results: Average spectra from edema exhibited higher water absorption than non-edema spectra. SWIR MCI differentiated healthy volunteers from a population representing all pitting edema grades with 97.1% accuracy (N = 103 shins). Additionally, SWIR MCI correctly classified shin pitting edema levels in patients with 81.6% accuracy. Conclusions: Our study successfully achieved the two primary endpoints. Application of SWIR MCI to monitor patients while actively receiving HF treatment is necessary to validate SWIR MCI as an HF monitoring technology.

Significance: Fourier ptychography microscopy (FPM) is a computational optical imaging technology that employs angularly varying illuminations and a phase retrieval algorithm to achieve a wide field of view and high-resolution imaging simultaneously. In the FPM, LED position error will reduce the quality of the reconstructed high-resolution image. To correct the LED positions, current methods consider each of the LED positions as independent and use an optimization algorithm to get each of the positions. When the positional misalignment is large or the search position falls into a local optimal value, the current methods may lack stability and accuracy. Aim: We improve the model of the LED position and propose an accurate and stable two-step correction scheme (tcFPM) to calibrate the LED position error. Approach: The improved LED positions model combines the overall offset, which represents the relative deviation of the LED array and the optical axis, with the slight deviation of each LED’s independent position. In the tcFPM, the overall offset of the LED array is corrected at first, which obtains an approximate value of the overall offset of the LED array. Then the position of each LED is precisely adjusted, which obtains the slight offset of each LED. Results: This LED position error model is more in line with the actual situation. The simulation and experimental results show that the method has high accuracy in correcting the LED position. Furthermore, the reconstruction process of tcFPM is more stable and significantly improves the quality of the reconstruction results, which is compared with some LED position error correction methods. Conclusions: An LED position error correction technology is proposed, which has a stable iterative process and improves the reconstruction accuracy of complex amplitude.

Significance: The Cherenkov emission spectrum overlaps with that of ambient room light sources. Choice of room lighting devices dramatically affects the efficient detection of Cherenkov emission during patient treatment. Aim: To determine optimal room light sources allowing Cherenkov emission imaging in normally lit radiotherapy treatment delivery rooms. Approach: A variety of commercial light sources and long-pass (LP) filters were surveyed for spectral band separation from the red to near-infrared Cherenkov light emitted by tissue. Their effects on signal-to-noise ratio (SNR), Cherenkov to background signal ratio, and image artifacts were quantified by imaging irradiated tissue equivalent phantoms with an intensified time-gated CMOS camera. Results: Because Cherenkov emission from tissue lies largely in the near-infrared spectrum, a controlled choice of ambient light that avoids this spectral band is ideal, along with a camera that is maximally sensitive to it. An RGB LED light source produced the best SNR out of all sources that mimic room light temperature. A 675-nm LP filter on the camera input further reduced ambient light detected (optical density > 3), achieving maximal SNR for Cherenkov emission near 40. Reduction of the room light signal reduced artifacts from specular reflection on the tissue surface and also minimized spurious Cherenkov signals from non-tissue features such as bolus. Conclusions: LP filtering during image acquisition for near-infrared light in tandem with narrow band LED illuminated rooms improves image quality, trading off the loss of red wavelengths for better removal of room light in the image. This spectral filtering is also critically important to remove specular reflection in the images and allow for imaging of Cherenkov emission through clear bolus. Beyond time-gated external beam therapy systems, the spectral separation methods can be utilized for background removal for continuous treatment delivery methods including proton pencil beam scanning systems and brachytherapy.

Significance: The scattering and polarization characteristics of various organs of in vivo wildtype zebrafish in three development stages were investigated using a non-destructive and label-free approach. The presented results showed a promising first step for the usability of Jones-matrix optical coherence tomography (JM-OCT) in zebrafish-based research. Aim: We aim to visualize and quantify the scatter and polarization signatures of various zebrafish organs for larvae, juvenile, and young adult animals in vivo in a non-invasive and label-free way. Approach: A custom-built polarization-sensitive JM-OCT setup in combination with a motorized translation stage was utilized to investigate live zebrafish. Depth-resolved scattering (intensity and attenuation coefficient) and polarization (birefringence and degree of polarization uniformity) properties were analyzed. OCT angiography (OCT-A) was utilized to investigate the vasculature label-free and non-destructively. Results: The scatter and polarization signatures of the zebrafish organs such as the eye, gills, and muscles were investigated. The attenuation coefficient and birefringence changes between 1- and 2-month-old animals were evaluated in selected organs. OCT-A revealed the vasculature of in vivo larvae and juvenile zebrafish in a label-free manner. Conclusions: JM-OCT offers a rapid, label-free, non-invasive, tissue specific, and three-dimensional imaging tool to investigate in vivo processes in zebrafish in various development stages.

Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Significance: A non-destructive technique for accurately characterizing the spatial distribution of optical properties of soft tissue membranes may give improved outcomes in many tissue engineering applications. Aim: This study aimed to develop a non-destructive macroscopic imaging technique that is sensitive to optical anisotropy, typical of fibrous components in soft tissue membranes, and can address some of the difficulties caused by the complex turbid nature of these tissues. Approach: A near-infrared Mueller matrix imaging polarimeter employing logarithm decomposition was developed and used to conduct transmission measurements of all the polarization properties across the full thickness of bovine pericardium tissue. Results: The full Mueller matrix was measured across a 70 mm×70 mm sample of calf bovine pericardium and revealed significant retardance (linear and circular) and depolarization in this tissue. Regions with a uniform axis of optical anisotropy were identified. Mueller matrix imaging demonstrated that the exhibited circular retardance was sufficient to lead to possible misinterpretation of apparent fiber orientation when using conventional polarization imaging techniques for such tissues. Conclusions: Mueller matrix imaging can identify regional distributions of optical anisotropy in calf bovine pericardium. This new capability is a promising development in non-destructive imaging for tissue selection.

Significance: An increasing interest in the area of biological effects at exposure of tissues and cells to the terahertz (THz) radiation is driven by a rapid progress in THz biophotonics, observed during the past decades. Despite the attractiveness of THz technology for medical diagnosis and therapy, there is still quite limited knowledge about safe limits of THz exposure. Different modes of THz exposure of tissues and cells, including continuous-wave versus pulsed radiation, various powers, and number and duration of exposure cycles, ought to be systematically studied. Aim: We provide an overview of recent research results in the area of biological effects at exposure of tissues and cells to THz waves. Approach: We start with a brief overview of general features of the THz-wave–tissue interactions, as well as modern THz emitters, with an emphasis on those that are reliable for studying the biological effects of THz waves. Then, we consider three levels of biological system organization, at which the exposure effects are considered: (i) solutions of biological molecules; (ii) cultures of cells, individual cells, and cell structures; and (iii) entire organs or organisms; special attention is devoted to the cellular level. We distinguish thermal and nonthermal mechanisms of THz-wave–cell interactions and discuss a problem of adequate estimation of the THz biological effects’ specificity. The problem of experimental data reproducibility, caused by rareness of the THz experimental setups and an absence of unitary protocols, is also considered. Results: The summarized data demonstrate the current stage of the research activity and knowledge about the THz exposure on living objects. Conclusions: This review helps the biomedical optics community to summarize up-to-date knowledge in the area of cell exposure to THz radiation, and paves the ways for the development of THz safety standards and THz therapeutic applications.

Significance: As linear array transducers are widely used in clinical ultrasound imaging, photoacoustic tomography (PAT) with linear arrays is similarly suitable for clinical applications. However, due to the limited-view problem, a linear array has limited performance and leads to artifacts and blurring, which has hindered its broader application. There is a need to address the limited-view problem in PAT imaging with linear arrays. Aim: We investigate potential approaches for improving PAT reconstruction from linear array, by optimizing the detection geometry and implementing iterative reconstruction. Approach: PAT imaging with a single-array, dual-probe configurations in parallel-shape and L-shape, and square-shape configuration are compared in simulations and phantom experiments. An iterative model-based algorithm based on the variance-reduced stochastic gradient descent (VR-SGD) method is implemented. The optimum configuration found in simulation is validated on phantom experiments. Results: PAT imaging with dual-probe detection and VR-SGD algorithm is found to improve the limited-view problem compared to a single probe and provide comparable performance as full-view geometry in simulation. This configuration is validated in experiments where more complete structure is obtained with reduced artifacts compared with a single array. Conclusions: PAT with dual-probe detection and iterative reconstruction is a promising solution to the limited-view problem of linear arrays.

Significance: The current gold standard for monitoring small intestinal transplant (IT) rejection is endoscopic visual assessment and biopsy of suspicious lesions; however, these lesions are only superficially visualized by endoscopy. Invasive biopsies provide a coarse sampling of tissue health without depicting the true presence and extent of any pathology. Optical coherence tomography (OCT) presents a potential alternative approach with significant advantages over traditional white-light endoscopy. Aim: The aim of our investigation was to evaluate OCT performance in distinguishing clinically relevant morphological features associated with IT graft failure. Approach: OCT was applied to evaluate the small bowel tissues of two rhesus macaques that had undergone IT of the ileum. The traditional assessment from routine histological observation was compared with OCT captured using a handheld surgical probe during the days post-transplant and subsequently was compared with histophaology. Results: The reported OCT system was capable of identifying major biological landmarks in healthy intestinal tissue. Following IT, one nonhuman primate (NHP) model suffered a severe graft ischemia, and the second NHP graft failed due to acute cellular rejection. OCT images show visual evidence of correspondence with histological signs of IT rejection. Conclusions: Results suggest that OCT imaging has significant potential to reveal morphological changes associated with IT rejection and to improve patient outcomes overall.

Significance: Sub-diffuse optical properties may serve as useful cancer biomarkers, and wide-field heatmaps of these properties could aid physicians in identifying cancerous tissue. Sub-diffuse spatial frequency domain imaging (sd-SFDI) can reveal such wide-field maps, but the current time cost of experimentally validated methods for rendering these heatmaps precludes this technology from potential real-time applications. Aim: Our study renders heatmaps of sub-diffuse optical properties from experimental sd-SFDI images in real time and reports these properties for cancerous and normal skin tissue subtypes. Approach: A phase function sampling method was used to simulate sd-SFDI spectra over a wide range of optical properties. A machine learning model trained on these simulations and tested on tissue phantoms was used to render sub-diffuse optical property heatmaps from sd-SFDI images of cancerous and normal skin tissue. Results: The model accurately rendered heatmaps from experimental sd-SFDI images in real time. In addition, heatmaps of a small number of tissue samples are presented to inform hypotheses on sub-diffuse optical property differences across skin tissue subtypes. Conclusion: These results bring the overall process of sd-SFDI a fundamental step closer to real-time speeds and set a foundation for future real-time medical applications of sd-SFDI such as image guided surgery.

Significance: Effective vein visualization is critically important for several clinical procedures, such as venous blood sampling and intravenous injection. Existing technologies using infrared device or ultrasound rely on professional equipment and are not suitable for daily medical care. A regression-based vein visualization method is proposed. Aim: We visualize veins from conventional RGB images to provide assistance in venipuncture procedures as well as clinical diagnosis of some venous insufficiency. Approach: The RGB images taken by digital cameras are first transformed to spectral reflectance images using Wiener estimation. Multiple regression analysis is then applied to derive the relationship between spectral reflectance and the concentrations of pigments. Monte Carlo simulation is adopted to get prior information. Finally, vein patterns are visualized from the spatial distribution of pigments. To minimize the effect of illumination on skin color, light correction and shading removal operations are performed in advance. Results: Experimental results from inner forearms of 60 subjects show the effectiveness of the regression-based method. Subjective and objective evaluations demonstrate that the clarity and completeness of vein patterns can be improved by light correction and shading removal. Conclusions: Vein patterns can be successfully visualized from RGB images without any professional equipment. The proposed method can assist in venipuncture procedures. It also shows promising potential to be used in clinical diagnosis and treatment of some venous insufficiency.

Significance: Optical imaging of Cherenkov emission during radiation therapy could be used to verify dose delivery in real-time if a more comprehensive quantitative understanding of the factors affecting emission intensity could be developed. Aim: This study aims to explore the change in diffuse Cherenkov emission intensity with x-ray beam energy from irradiated tissue, both theoretically and experimentally. Approach: Derivation of the emitted Cherenkov signal was achieved using diffusion theory, and experimental studies with 6 to 18 MV energy x-rays were performed in tissue phantoms to confirm the model predictions as related to the radiation build-up factor with depth into tissue. Results: Irradiation at lower x-ray energies results in a greater surface dose and higher build-up slope, which results in a ∼46% greater diffusely emitted Cherenkov signal per unit dose at 6 MV relative to 18 MV x-rays. However, this phenomenon competes with a decrease in signal from less Cherenkov photons being generated at lower energies, a ∼44% reduction at 6 versus 18 MV. The result is an emitted Cherenkov signal that is nearly constant with beam energy. Conclusions: This study explains why the observed Cherenkov emission from tissue is not a strong function of beam energy, despite the known strong correlation between Cherenkov intensity and particle energy in the absence of build-up and scattering effects.

Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

Significance: Photoacoustic imaging (PAI) is a powerful emerging technology with broad clinical applications, but consensus test methods are needed to standardize performance evaluation and accelerate translation. Aim: To review consensus image quality test methods for mature imaging modalities [ultrasound, magnetic resonance imaging (MRI), x-ray CT, and x-ray mammography], identify best practices in phantom design and testing procedures, and compare against current practices in PAI phantom testing. Approach: We reviewed scientific papers, international standards, clinical accreditation guidelines, and professional society recommendations describing medical image quality test methods. Observations are organized by image quality characteristics (IQCs), including spatial resolution, geometric accuracy, imaging depth, uniformity, sensitivity, low-contrast detectability, and artifacts. Results: Consensus documents typically prescribed phantom geometry and material property requirements, as well as specific data acquisition and analysis protocols to optimize test consistency and reproducibility. While these documents considered a wide array of IQCs, reported PAI phantom testing focused heavily on in-plane resolution, depth of visualization, and sensitivity. Understudied IQCs that merit further consideration include out-of-plane resolution, geometric accuracy, uniformity, low-contrast detectability, and co-registration accuracy. Conclusions: Available medical image quality standards provide a blueprint for establishing consensus best practices for photoacoustic image quality assessment and thus hastening PAI technology advancement, translation, and clinical adoption.

Significance: Artificial skin (AS) is widely used in dermatology, pharmacology, and toxicology, and has great potential in transplant medicine, burn wound care, and chronic wound treatment. There is a great demand for high-quality AS product and a non-invasive detection method is highly desirable. Aim: To quantify the constructure parameters (i.e., thickness and surface roughness) of AS samples in the culture cycle and explore the growth regularities using optical coherent tomography (OCT). Approach: An adaptive interface detection algorithm is developed to recognize surface points in each A-scan, offering a rapid method to calculate parameters without constructing OCT B-scan pictures and further achieving realizing real-time quantification of AS thickness and surface roughness. Experiments on standard roughness plates and H&E-staining microscopy were performed as a verification. Results: As applied on the whole cycle of AS culture, our method’s results show that during the air–liquid culture, the surface roughness of the skin first decreases and then exhibits an increase, which implies coincidence with the degree of keratinization under a microscope. And normal and typical abnormal samples can be differentiated by thickness and roughness parameters during the culture cycle. Conclusions: The adaptive interface detection algorithm is suitable for high-sensitivity, fast detection, and quantification of the interface with layered characteristic tissues, and can be used for non-destructive detection of the growth regularity of AS sample thickness and roughness during the culture cycle.

Significance: Imaging biofluid flow under physiologic conditions aids in understanding disease processes and health complications. We present a method employing a microparallel plate strain induction chamber (MPPSIC) amenable to optical coherence tomography to track depth-resolved lateral displacement in fluids in real time while under constant and sinusoidal shear. Aim: Our objective is to track biofluid motion under shearing conditions found in the respiratory epithelium, first validating methods in Newtonian fluids and subsequently assessing the capability of motion-tracking in bronchial mucus. Approach: The motion of polystyrene microspheres in aqueous glycerol is tracked under constant and sinusoidal applied shear rates in the MPPSIC and is compared with theory. Then 1.5 wt. % bronchial mucus samples considered to be in a normal hydrated state are studied under sinusoidal shear rates of amplitudes 0.7 to 3.2 s−1. Results: Newtonian fluids under low Reynolds conditions (Re∼10−4) exhibit velocity decreases directly proportional to the distance from the plate driven at both constant and oscillating velocities, consistent with Navier–Stokes’s first and second problems at finite depths. A 1.5 wt. % mucus sample also exhibits a uniform shear strain profile. Conclusions: The MPPSIC provides a new capability for studying biofluids, such as mucus, to assess potentially non-linear or strain-rate-dependent properties in a regime that is relevant to the mucus layer in the lung epithelium.

Significance: Hyperspectral imaging (HSI) has emerged as a promising optical technique. Besides optical properties of a sample, other sample physical properties also affect the recorded images. They are significantly affected by the sample curvature and sample surface to camera distance. A correction method to reduce the artifacts is necessary to reliably extract sample properties. Aim: Our aim is to correct hyperspectral images using the three-dimensional (3D) surface data and assess how the correction affects the extracted sample properties. Approach: We propose the combination of HSI and 3D profilometry to correct the images using the Lambert cosine law. The feasibility of the correction method is presented first on hemispherical tissue phantoms and next on human hands before, during, and after the vascular occlusion test (VOT). Results: Seven different phantoms with known optical properties were created and imaged with a hyperspectral system. The correction method worked up to 60 deg inclination angle, whereas for uncorrected images the maximum angles were 20 deg. Imaging hands before, during, and after VOT shows good agreement between the expected and extracted skin physiological parameters. Conclusions: The correction method was successfully applied on the images of tissue phantoms of known optical properties and geometry and VOT. The proposed method could be applied to any reflectance optical imaging technique and should be used whenever the sample parameters need to be extracted from a curved surface sample.

Significance: Polyp size is important for selecting the surveillance interval or treatment policy. Nevertheless, it is challenging to accurately estimate the polyp size during endoscopy. An easy and cost-effective function to assist in polyp size estimation is required. Aim: To propose a virtual scale function for endoscopy and evaluate its performance and expected accuracy. Approach: An adaptive virtual scale behavior was demonstrated. The measurement error of the virtual scale along the distance between the tip of the endoscope and the object plane was evaluated using graph paper. The accuracy of polyp size estimation by an expert endoscopist was compared with the accuracy of the biopsy forceps method using phantom images. Results: The measurement errors of the virtual scale were ≤ 0.7 mm when the distance to the graph paper, which faced the tip of the endoscope, varied from 4 to 30 mm. The accuracy with the virtual scale was significantly higher than that obtained with biopsy forceps (5.3 ± 5.5 % versus 11.9 ± 9.4 % , P

Significance: Performance of an acousto-optic imaging system is limited by light fluence rate and acoustic pressure field distributions characteristics. In optically scattering media, the former determines the achievable contrast, whereas the latter the imaging resolution. The system parameters can be shaped by changing relative positions of ultrasound (US) transducer array and optodes. However, in the case of many potential clinical applications, optimization possibilities in this regard are limited, as a sample is accessible from one side only and using a water tank for coupling is not feasible. Aim: We investigate the possibilities of improving performance of an acousto-optic imaging system operating in reflection mode geometry with linear US array in direct contact with a sample using plane wave instead of focused US pulses. Approach: Differences in acoustic pressure field distributions for various transducer excitation patterns were determined numerically and experimentally. Acousto-optic images of phantoms with and without optically absorbing inclusions were acquired by measuring laser speckle contrast decrease due to the light modulation by plane wave and focused US pulses with different apodization patterns. Results: The residual acoustic pressure field components occupy relatively large volume and contribute to light modulation. Using nonsteered plane wave US pulses instead of focused ones allows one to mitigate their influence. It also allows one to obtain clear two-dimensional reconstructions of light fluence rate maps by shifting transducer apodization along the lateral direction. Conclusions: Using nonsteered plane wave US pulses allows one to achieve better imaging performance than with focused pulses in the assumed system geometry.

Significance: We introduce a model for better calibration of the trapping force using an equal but oppositely directed drag force acting on a trapped red blood cell (RBC). We demonstrate this approach by studying RBCs’ elastic properties from deidentified sickle cell anemia (SCA) and sickle cell trait (SCT) blood samples. Aim: A laser trapping (LT) force was formulated and analytically calculated in a cylindrical model. Using this trapping force relative percent difference, the maximum (longitudinal) and minimum (transverse) radius rate and stiffness were used to study the elasticity. Approach: The elastic property of SCA and SCT RBCs was analyzed using LT technique with computer controlled piezo-driven stage, in order to trap and stretch the RBCs. Results: For all parameters, the results show that the SCT RBC samples have higher elastic property than the SCA RBCs. The higher rigidity in the SCA cell may be due to the lipid composition of the membrane, which was affected by the cholesterol concentration. Conclusions: By developing a theoretical model for different trapping forces, we have also studied the elasticity of RBCs in SCT (with hemoglobin type HbAS) and in SCA (with hemoglobin type HbSS). The results for the quantities describing the elasticity of the cells consistently showed that the RBCs in the SCT display lower rigidity and higher deformability than the RBCs with SCA.

Significance: Our work demonstrates in preclinical models that continuous-wave transrectal diffuse optical tomography (TRDOT) can be used to accurately monitor photothermal therapy (PTT) and, in particular, the progression of the photocoagulation boundary toward the rectum. When used in patients, this should prevent rectal damage during PTT, thereby achieving maximum treatment efficacy while ensuring safety, using a technology platform suitable for wide dissemination. Aim: We aim to validate that TRDOT measurements analyzed using a shape-based image-reconstruction algorithm (SBDOT) allow localization of the photocoagulation boundary during PTT within ±1 mm toward the rectum in the transverse plane. Approach: TRDOT measurements were performed in tissue-simulating phantoms, ex vivo tissues, and an in vivo canine prostate model. The accuracy and sensitivity of reconstructing the size and location of the coagulation zone were determined, based on changes in the tissue absorption and reduced scattering coefficients upon photocoagulation. The reconstruction also yields the native and coagulated tissue optical properties. Results: The TRDOT measurements and SBDOT reconstruction algorithm were confirmed to perform sufficiently well for clinical translation in PTT monitoring, recovering the location of the coagulation boundary within ±1 mm compared to the true value as determined by direct visualization postexcision and/or MRI. Conclusions: Implementing previously described TRDOT instrumentation and SBDOT image reconstruction in different tissue models confirms the potential for clinincal translation, including required refinements of the system and reconstruction algorithm.

SignificanceSpeckle variations induced by intracellular motion (IM) in the urothelium was observed in optical coherence tomography (OCT) images. It is feasible to use the IM as a dynamic contrast to segment the urothelium with only two sequential OCT images. This new method opens the possibility of tracking the distribution of the urothelial cells to identify the microinvasion of bladder tumors.ApproachWith fresh porcine bladder tissue, IM was analyzed by tracking speckle variations using autocorrelation function, then quantified with CONTINE algorism to identify the decorrelation time (DT) of the speckle variations. Variance analysis is conducted to show IM amplitude and distribution in the urothelium. The segmentation of the urothelium is demonstrated using tissue samples with and without significantly stretching.ResultsSignificant speckle variations induced by IM exists in the urothelium. However, the distribution of the IM is heterogeneous. The DTs are majorly distributed between 1ms to 30ms. Using the IM as a dynamic contrast, the urothelium can be accurately and exclusively segmented, even the layer structure of the bladder is invisible.ConclusionsWith fresh porcine bladders, we show that IM can be used as a dynamic contrast to exclusively segment the urothelium. This contrast may provide a new mechanism for OCT to diagnosis the invasion of urothelial cancerous cells for the better staging of bladder cancer.

Raman spectroscopy has shown very promising results in medical diagnostics by providing label-free and highly specific molecular information of pathological tissue ex vivo and in vivo. Nevertheless, the high specificity of Raman spectroscopy comes at a price, i.e., low acquisition rate, no direct access to depth information, and limited sampling areas. However, a similar case regarding advantages and disadvantages can also be made for other highly regarded optical modalities, such as optical coherence tomography, autofluorescence imaging and fluorescence spectroscopy, fluorescence lifetime microscopy, second-harmonic generation, and others. While in these modalities the acquisition speed is significantly higher, they have no or only limited molecular specificity and are only sensitive to a small group of molecules. It can be safely stated that a single modality provides only a limited view on a specific aspect of a biological specimen and cannot assess the entire complexity of a sample. To solve this issue, multimodal optical systems, which combine different optical modalities tailored to a particular need, become more and more common in translational research and will be indispensable diagnostic tools in clinical pathology in the near future. These systems can assess different and partially complementary aspects of a sample and provide a distinct set of independent biomarkers. Here, we want to give an overview on the development of multimodal systems that use RS in combination with other optical modalities to improve the diagnostic performance.

Significance: The morphological properties and hemoglobin (Hb) content of red blood cells (RBCs) are essential biomarkers to diagnose or monitor various types of hematological disorders. Label-free mass mapping approaches enable accurate Hb quantification from individual cells, serving as promising alternatives to conventional hematology analyzers. Deep ultraviolet (UV) microscopy is one such technique that allows high-resolution, molecular imaging, and absorption-based mass mapping. Aim: To compare UV absorption-based mass mapping at four UV wavelengths and understand variations across wavelengths and any assumptions necessary for accurate Hb quantification. Approach: Whole blood smears are imaged with a multispectral UV microscopy system, and the RBCs’ dry masses are computed. This approach is compared to quantitative phase imaging-based mass mapping using data from an interferometric UV imaging system. Results: Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm. Conclusions: A full hematological analysis (including white blood cell identification and characterization, and Hb quantification) may be achieved using a single UV illumination wavelength, thereby improving the speed and cost-effectiveness.

Significance: Orthopedic surgery currently comprises over 1.5 million cases annually in the United States alone and is growing rapidly with aging populations. Emerging optical sensing techniques promise fewer side effects with new, more effective approaches aimed at improving patient outcomes following orthopedic surgery. Aim: The aim of this perspective paper is to outline potential applications where fiberoptic-based approaches can complement ongoing development of minimally invasive surgical procedures for use in orthopedic applications. Approach: Several procedures involving orthopedic and spinal surgery, along with the clinical challenge associated with each, are considered. The current and potential applications of optical sensing within these procedures are discussed and future opportunities, challenges, and competing technologies are presented for each surgical application. Results: Strong research efforts involving sensor miniaturization and integration of optics into existing surgical devices, including K-wires and cranial perforators, provided the impetus for this perspective analysis. These advances have made it possible to envision a next-generation set of devices that can be rigorously evaluated in controlled clinical trials to become routine tools for orthopedic surgery. Conclusions: Integration of optical devices into surgical drills and burrs to discern bone/tissue interfaces could be used to reduce complication rates across a spectrum of orthopedic surgery procedures or to aid less-experienced surgeons in complex techniques, such as laminoplasty or osteotomy. These developments present both opportunities and challenges for the biomedical optics community.

Significance: Quantitative stiffness information can be a powerful aid for tumor or fibrosis diagnosis. Currently, very promising elastography approaches developed for non-contact biomedical imaging are based on transient shear-waves imaging. Transient elastography offers quantitative stiffness information by tracking the propagation of a wave front. The most common method used to compute stiffness from the acquired propagation movie is based on shear-wave time-of-flight calculations. Aim: We introduce an approach to transient shear-wave elastography with spatially coherent sources, able to yield full-field quantitative stiffness maps with reduced artifacts compared to typical artifacts observed in time-of-flight. Approach: A noise-correlation algorithm developed for passive elastography is adapted to spatially coherent narrow or any band sources. This noise-correlation-inspired (NCi) method is employed in parallel with a classic time-of-flight approach. Testing is done on simulation images, experimental validation is conducted with a digital holography setup on controlled homogeneous samples, and full-field quantitative stiffness maps are presented for heterogeneous samples and ex-vivo biological tissues. Results: The NCi approach is first validated on simulations images. Stiffness images processed by the NCi approach on simulated inclusions display significantly less artifacts than with a time-of-flight reconstruction. The adaptability of the NCi algorithm to narrow or any band shear-wave sources was tested successfully. Experimental testing on homogeneous samples demonstrates similar values for both the time-of-flight and the NCi approach. Soft inclusions in agarose sample could be resolved using the NCi method and feasibility on ex-vivo biological tissues is presented. Conclusions: The presented NCi approach was successful in computing quantitative full-field stiffness maps with narrow and broadband source signals on simulation and experimental images from a digital holography setup. Results in heterogeneous media show that the NCi approach could provide stiffness maps with less artifacts than with time-of-flight, demonstrating that a NCi algorithm is a promising approach for shear-wave transient elastography with spatially coherent sources.

Significance: Spatial frequency domain imaging (SFDI) is a wide-field imaging technique that provides quantitative maps of tissue optical properties. We describe a compact SFDI imager that employs a multispectral compound-eye camera. This design enables simultaneous image acquisition at multiple wavelengths. Such a device has potential for application for quantitative evaluation of superficial tissues by nonspecialists in low-resource settings. Aim: The aim of this work was to develop a compact SFDI imager for widefield imaging of in-vivo tissue optical properties and verify its ability to measure optical properties of tissue-simulating phantoms and in a preclinical model of burn wounds. Approach: This compound-eye imager was constructed using a CMOS sensor subdivided into multiple regions, each having a bandpass filter and objective lens. The ability of the instrument to image optical properties was compared with (1) a commercial SFDI imager and (2) a laboratory-based system. Initial validation of ability to accurately characterize optical properties was performed using a tissue-simulating optical phantom. It was then applied to an established murine model of thermal contact burn severity. In-vivo measurements of the optical properties of rat skin were performed before and after the application of burns. Histology was used to verify burn severity. Results: Measurements of the tissue-simulating phantom optical properties made using the compound-eye imager agree with measurements made using the two comparison SFDI devices. For the murine burn model, the burns showed a decrease in the reduced scattering coefficient at all measurement wavelengths compared with preburn measurements at the same locations. This is consistent with previously reported changes in scattering that occur in full-thickness burns. Conclusion: We demonstrate the potential for SFDI to be translated into compact form factor using a compound-eye camera that is capable of obtaining multiple wavelengths channels simultaneously.

Significance: Diffuse optical spectroscopic imaging (DOSI) is a versatile technology sensitive to changes in tissue composition and hemodynamics and has been used for a wide variety of clinical applications. Specific applications have prompted the development of versions of the DOSI technology to fit specific clinical needs. This work describes the development and characterization of a multi-modal DOSI (MM-DOSI) system that can acquire metabolic, compositional, and pulsatile information at multiple penetration depths in a single hardware platform. Additionally, a 3D tracking system is integrated with MM-DOSI, which enables registration of the acquired data to the physical imaging area. Aim: We demonstrate imaging, layered compositional analysis, and metabolism tracking capabilities using a single MM-DOSI system on optical phantoms as well as in vivo human tissue. Approach: We characterize system performance with a silicone phantom containing an embedded object. To demonstrate multi-layer sensitivity, we imaged human calf tissue with a 4.8-mm skin-adipose thickness. Human thenar tissue was also measured using a combined broadband DOSI and continuous-wave near-infrared spectroscopy method (∼15 Hz acquisition rate). Results: High-resolution optical property maps of absorption (μa) and reduced scattering (μs ′ ) were recovered on the phantom by capturing over 1000 measurement points in under 5 minutes. On human calf tissue, we show two probing depth layers have significantly different (p

Significance: Screening and early detection of oral potentially malignant lesions (OPMLs) are of great significance in reducing the mortality rates associated with head and neck malignancies. Intra-oral multispectral optical imaging of tissues in conjunction with cloud-based machine learning (CBML) can be used to detect oral precancers at the point-of-care (POC) and guide the clinician to the most malignant site for biopsy. Aim: Develop a bimodal multispectral imaging system (BMIS) combining tissue autofluorescence and diffuse reflectance (DR) for mapping changes in oxygenated hemoglobin (HbO2) absorption in the oral mucosa, quantifying tissue abnormalities, and guiding biopsies. Approach: The hand-held widefield BMIS consisting of LEDs emitting at 405, 545, 575, and 610 nm, 5MPx monochrome camera, and proprietary Windows-based software was developed for image capture, processing, and analytics. The DR image ratio (R610/R545) was compared with pathologic classification to develop a CBML algorithm for real-time assessment of tissue status at the POC. Results: Sensitivity of 97.5% and specificity of 92.5% were achieved for discrimination of OPML from patient normal in 40 sites, whereas 82% sensitivity and 96.6% specificity were obtained for discrimination of abnormal (OPML + SCC) in 89 sites. Site-specific algorithms derived for buccal mucosa (27 sites) showed improved sensitivity and specificity of 96.3% for discrimination of OPML from normal. Conclusions: Assessment of oral cancer risk is possible by mapping of HbO2 absorption in tissues, and the BMIS system developed appears to be suitable for biopsy guidance and early detection of oral cancers.