Thanks to reviewers who served JBO in 2021.

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: 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: 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: 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: 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: 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: 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: 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: 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 τ.

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 τ cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present.

Significance: 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.

Aim: The purpose of this study is to investigate the feasibility of utilizing FPM to reconstruct high-resolution chromosome images.

Approach: 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.

Results: 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.

Conclusions: This study initially verified that it is feasible to utilize FPM to develop a high-resolution and wide-field chromosome sample scanner.

Significance: Performance improvements in microfluidic systems depend on accurate measurement and fluid control on the micro- and nanoscales. New applications are continuously leading to lower volumetric flow rates.

Aim: We focus on improving an optofluidic system for measuring and calibrating microflows to the sub-nanoliter per minute range.

Approach: Measurements rely on an optofluidic system that delivers excitation light and records fluorescence in a precise interrogation region of a microfluidic channel. Exploiting a scaling relationship between the flow rate and fluorescence emission after photobleaching, the system enables real-time determination of flow rates.

Results: Here, we demonstrate improved calibration of a flow controller to 1% uncertainty. Further, the resolution of the optofluidic flow meter improved to less than 1  nL  /  min with 5% uncertainty using a molecule with a 14-fold smaller diffusion coefficient than our previous report.

Conclusions: We demonstrate new capabilities in sub-nanoliter per minute flow control and measurement that are generalizable to cutting-edge light-material interaction and molecular diffusion for chemical and biomedical industries.

Significance: Pseudomonas (P.) aeruginosa, a common cause of infection in burns, acquires antibiotic resistance easily and forms biofilms efficiently. Thus, it is difficult to control P. aeruginosa infection in burn wounds, which causes lethal septicemia. Antimicrobial photodynamic therapy (aPDT) is attractive as a new strategy to treat burn wound infections with drug-resistant bacteria.

Aim: We examined the efficacy of methylene blue (MB)-mediated aPDT with various additives in a tissue depth-resolved manner to find conditions that minimize the bacterial invasion.

Approach: We applied MB-mediated aPDT with LED array illumination to an extensive, full-thickness burn infected with P. aeruginosa in rats for three consecutive days (days 0, 1, and 2). On day 2, the depth distributions of bacteria were assessed based on the histological analysis using Gram staining. We examined how the addition of ethylenediaminetetraacetic acid (EDTA), ethanol, and dimethyl sulfoxide (DMSO) affected the efficacy of aPDT.

Results: Pure MB-mediated aPDT significantly reduced the numbers of bacteria with biofilms on the wound surface and in the epidermis compared with those for the control tissue (saline only). However, there were many bacteria in the deeper region of the tissue. In contrast, MB/EDTA/ethanol/DMSO-mediated aPDT minimized the numbers of bacteria in the broad depth region of the tissue. Still, a limited number of bacteria was observed in the subcutaneous tissue.

Conclusions: The depthwise analysis of bacteria demonstrated the efficacy of the MB-mediated aPDT with the addition of EDTA, ethanol, and DMSO in controlling burn wound infections. However, further improvement of the therapy is needed to suppress bacterial migration into the deep tissue completely.