Significance: Extremity injury represents the leading cause of trauma hospitalizations among adults under the age of 65 years, and long-term impairments are often substantial. Restoring function depends, in large part, on bone and soft tissue healing. Thus, decisions around treatment strategy are based on assessment of the healing potential of injured bone and/or soft tissue. However, at the present, this assessment is based on subjective clinical clues and/or cadaveric studies without any objective measure. Optical imaging is an ideal method to solve several of these issues.

Aim: The aim is to highlight the current challenges in assessing bone and tissue perfusion/viability and the potentially high impact applications for optical imaging in orthopaedic surgery.

Approach: The prospective will review the current challenges faced by the orthopaedic surgeon and briefly discuss optical imaging tools that have been published. With this in mind, it will suggest key research areas that could be evolved to help make surgical assessments more objective and quantitative.

Results: Orthopaedic surgical procedures should benefit from incorporation of methods to measure functional blood perfusion or tissue metabolism. The types of measurements though can vary in the depth of tissue sampled, with some being quite superficial and others sensing several millimeters into the tissue. Most of these intrasurgical imaging tools represent an ideal way to improve surgical treatment of orthopaedic injuries due to their inherent point-of-care use and their compatibility with real-time management.

Conclusion: While there are several optical measurements to directly measure bone function, the choice of tools can determine also the signal strength and depth of sampling. For orthopaedic surgery, real-time data regarding bone and tissue perfusion should lead to more effective patient-specific management of common orthopaedic conditions, requiring deeper penetrance commonly seen with indocyanine green imaging. This will lower morbidity and result in decreased variability associated with how these conditions are managed.

Significance: Spatial frequency domain imaging (SFDI) is a quantitative imaging method to measure absorption and scattering of tissue, from which several chromophore concentrations (e.g., oxy-/deoxy-/meth-hemoglobin, melanin, and carotenoids) can be calculated. Employing a method to extract additional spectral bands from RGB components (that we named cross-channels), we designed a handheld SFDI device to account for these pigments, using low-cost, consumer-grade components for its implementation and characterization.

Aim: With only three broad spectral bands (red, green, blue, or RGB), consumer-grade devices are often too limited. We present a methodology to increase the number of spectral bands in SFDI devices that use RGB components without hardware modification.

Approach: We developed a compact low-cost RGB spectral imager using a color CMOS camera and LED-based mini projector. The components’ spectral properties were characterized and additional cross-channel bands were calculated. An alternative characterization procedure was also developed that makes use of low-cost equipment, and its results were compared. The device performance was evaluated by measurements on tissue-simulating optical phantoms and in-vivo tissue. The measurements were compared with another quantitative spectroscopy method: spatial frequency domain spectroscopy (SFDS).

Results: Out of six possible cross-channel bands, two were evaluated to be suitable for our application and were fully characterized (520  ±  20  nm; 556  ±  18  nm). The other four cross-channels presented a too low signal-to-noise ratio for this implementation. In estimating the optical properties of optical phantoms, the SFDI data have a strong linear correlation with the SFDS data (R²  =  0.987, RMSE  =  0.006 for μ_a, R²  =  0.994, RMSE  =  0.078 for μs′).

Conclusions: We extracted two additional spectral bands from a commercial RGB system at no cost. There was good agreement between our device and the research-grade SFDS system. The alternative characterization procedure we have presented allowed us to measure the spectral features of the system with an accuracy comparable to standard laboratory equipment.

Significance: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O₂ instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production.

Aim: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells.

Approach: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light.

Results: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate.

Conclusions: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

Significance: Indirect imaging problems in biomedical optics generally require repeated evaluation of forward models of radiative transport, for which Monte Carlo is accurate yet computationally costly. We develop an approach to reduce this bottleneck, which has significant implications for quantitative tomographic imaging in a variety of medical and industrial applications.

Aim: Our aim is to enable computationally efficient image reconstruction in (hybrid) diffuse optical modalities using stochastic forward models.

Approach: Using Monte Carlo, we compute a fully stochastic gradient of an objective function for a given imaging problem. Leveraging techniques from the machine learning community, we then adaptively control the accuracy of this gradient throughout the iterative inversion scheme to substantially reduce computational resources at each step.

Results: For example problems of quantitative photoacoustic tomography and ultrasound-modulated optical tomography, we demonstrate that solutions are attainable using a total computational expense that is comparable to (or less than) that which is required for a single high-accuracy forward run of the same Monte Carlo model.

Conclusions: This approach demonstrates significant computational savings when approaching the full nonlinear inverse problem of optical property estimation using stochastic methods.

Significance: Two-dimensional (2-D) fully convolutional neural networks have been shown capable of producing maps of sO₂ from 2-D simulated images of simple tissue models. However, their potential to produce accurate estimates in vivo is uncertain as they are limited by the 2-D nature of the training data when the problem is inherently three-dimensional (3-D), and they have not been tested with realistic images.

Aim: To demonstrate the capability of deep neural networks to process whole 3-D images and output 3-D maps of vascular sO₂ from realistic tissue models/images.

Approach: Two separate fully convolutional neural networks were trained to produce 3-D maps of vascular blood oxygen saturation and vessel positions from multiwavelength simulated images of tissue models.

Results: The mean of the absolute difference between the true mean vessel sO₂ and the network output for 40 examples was 4.4% and the standard deviation was 4.5%.

Conclusions: 3-D fully convolutional networks were shown capable of producing accurate sO₂ maps using the full extent of spatial information contained within 3-D images generated under conditions mimicking real imaging scenarios. We demonstrate that networks can cope with some of the confounding effects present in real images such as limited-view artifacts and have the potential to produce accurate estimates in vivo.

Significance: Understanding how the valveless embryonic heart pumps blood is essential to elucidate biomechanical cues regulating cardiogenesis, which is important for the advancement of congenital heart defects research. However, methods capable of embryonic cardiac pumping analysis remain limited, and assessing this highly dynamic process in mammalian embryos is challenging. New approaches are critically needed to address this hurdle.

Aim: We report an imaging-based approach for functional assessment of localized pumping dynamics in the early tubular embryonic mouse heart.

Approach: Four-dimensional optical coherence tomography was used to obtain structural and Doppler hemodynamic imaging of the beating heart in live mouse embryos at embryonic day 9.25. The pumping assessment was performed based on the volumetric blood flow rate, flow resistance within the heart tube, and pressure gradient induced by heart wall movements. The relation between the blood flow, the pressure gradient, and the resistance to flow were evaluated through temporal analyses and Granger causality test.

Results: In the ventricles, our method revealed connections between the temporal profiles of pressure gradient and volumetric blood flow rate. Statistically significant causal relation from the pressure gradient to the blood flow was demonstrated. Our analysis also suggests that cardiac pumping in the early ventricles is a combination of suction and pushing. In contrast, in the outflow tract, where the conduction wave is slower than the blood flow, we did not find significant causal relation from pressure to flow, suggesting that, different from ventricular regions, the local active contraction of the outflow tract is unlikely to drive the flow in that region.

Conclusions: We present an imaging-based approach that enables localized assessment of pumping dynamics in the mouse tubular embryonic heart. This method creates a new opportunity for functional analysis of the pumping mechanism underlying the developing mammalian heart at early stages and could be useful for studying biomechanical changes in mutant embryonic hearts that model congenital heart defects.

Significance: Diffuse optical spectroscopic imaging (DOSI) measures quantitative functional and molecular information in thick tissue in a noninvasive manner using near-infrared light. DOSI may be useful for diagnosis and prognosis of bone pathologies including osteosarcoma and Ewing’s sarcoma, but little is currently known about DOSI-derived parameters in bony anatomic locations where this disease occurs.

Aim: Our goal is to quantify the optical characteristics and chromophore content of bony anatomic locations of healthy volunteers and assess differences due to anatomic region, age, sex, ethnicity, race, and body fat.

Approach: Fifty-five healthy volunteers aged 4 to 72 were enrolled in the study. The optical properties and quantitative tissue concentrations of oxyhemoglobin, deoxyhemoglobin, water, and lipids were assessed at the distal humerus, distal femur, and proximal tibia. Body fat was assessed using skinfold calipers. One volunteer underwent a more comprehensive body scan from neck to foot to explore chromophore distributions within an individual. Regression analysis was used to identify the most important sources of variation in the measured data set.

Results: Statistical differences between bony locations were found for scattering, water, and lipids, but not for hemoglobin. All chromophores had statistical differences with sex, but there were no significant age-related correlations. Regression analysis revealed that body fat measured with skinfold calipers was the most important predictor of oxy-, deoxy-, total hemoglobin, and lipids. Hemoglobin and lipid levels were highly correlated (ρ  ≥  0.7) over the subject population and within the single-subject body scan.

Conclusions: DOSI can successfully measure bony anatomic sites where osteosarcomas and Ewing’s sarcomas commonly occur. Future studies of bone pathology using DOSI should account for the variation caused by anatomic region, sex, race and ethnicity, and body fat as these cause substantial variations in DOSI-derived metrics.

Significance: Fluorescence polarization (FP) and fluorescence anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug–protein or protein–protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors.

Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy.

Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio.

Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of fluorescence intensities.

Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.

Significance: Hyperspectral imaging (HSI) can support intraoperative perfusion assessment, the identification of tissue structures, and the detection of cancerous lesions. The practical use of HSI for minimal-invasive surgery is currently limited, for example, due to long acquisition times, missing video, or large set-ups.

Aim: An HSI laparoscope is described and evaluated to address the requirements for clinical use and high-resolution spectral imaging.

Approach: Reflectance measurements with reference objects and resected human tissue from 500 to 1000 nm are performed to show the consistency with an approved medical HSI device for open surgery. Varying object distances are investigated, and the signal-to-noise ratio (SNR) is determined for different light sources.

Results: The handheld design enables real-time processing and visualization of HSI data during acquisition within 4.6 s. A color video is provided simultaneously and can be augmented with spectral information from push-broom imaging. The reflectance data from the HSI system for open surgery at 50 cm and the HSI laparoscope are consistent for object distances up to 10 cm. A standard rigid laparoscope in combination with a customized LED light source resulted in a mean SNR of 30 to 43 dB (500 to 950 nm).

Conclusions: Compact and rapid HSI with a high spatial- and spectral-resolution is feasible in clinical practice. Our work may support future studies on minimally invasive HSI to reduce intra- and postoperative complications.

Significance: Fluorocoxib D, N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide, is a water-soluble optical imaging agent to detect cyclooxygenase-2 (COX-2)-expressing cancer cells.

Aim: We evaluated the pharmacokinetic and safety properties of fluorocoxib D and its ability to detect cancer cells in vitro and in vivo.

Approach: Pharmacokinetic parameters of fluorocoxib D were assessed from plasma collected at designated time points after intravenous administration of 1  mg  /  kg fluorocoxib D in six research dogs using a high-performance liquid chromatography analysis. Safety of fluorocoxib D was assessed for 3 days after its administration using physical assessment, complete blood count, serum chemistry profile, and complete urinalysis in six research dogs. The ability of fluorocoxib D to detect COX-2-expressing cancer cells was performed using human 5637 cells in vitro and during rhinoscopy evaluation of specific fluorocoxib D uptake by canine cancer cells in vivo.

Results: No evidence of toxicity and no clinically relevant adverse events were noted in dogs. Peak concentration of fluorocoxib D (114.8  ±  50.5  ng  /  ml) was detected in plasma collected at 0.5 h after its administration. Pretreatment of celecoxib blocked specific uptake of fluorocoxib D in COX-2-expressing human 5637 cancer cells. Fluorocoxib D uptake was detected in histology-confirmed COX-2-expressing head and neck cancer during rhinoscopy in a client-owned dog in vivo. Specific tumor-to-normal tissue ratio of detected fluorocoxib D signal was in an average of 3.7  ±  0.9 using Image J analysis.

Conclusions: Our results suggest that fluorocoxib D is a safe optical imaging agent used for detection of COX-2-expressing cancers and their margins during image-guided minimally invasive biopsy and surgical procedures.

Significance: The hallmarks of digital holographic microscopy (DHM) compared with other quantitative phase imaging (QPI) methods are high speed, accuracy, spatial resolution, temporal stability, and polarization-sensitivity (PS) capability. The above features make DHM suitable for real-time quantitative PS phase imaging in a broad number of biological applications aimed at understanding cell growth and dynamic changes occurring during physiological processes and/or in response to pharmaceutical agents.

Aim: The insertion of a Fresnel biprism (FB) in the image space of a light microscope potentially turns any commercial system into a DHM system enabling QPI with the five desired features in QPI simultaneously: high temporal sensitivity, high speed, high accuracy, high spatial resolution, and PS. To the best of our knowledge, this is the first FB-based DHM system providing these five features all together.

Approach: The performance of the proposed system was calibrated with a benchmark phase object. The PS capability has been verified by imaging human U87 glioblastoma cells.

Results: The proposed FB-based DHM system provides accurate phase images with high spatial resolution. The temporal stability of our system is in the order of a few nanometers, enabling live-cell studies. Finally, the distinctive behavior of the cells at different polarization angles (e.g., PS capability) can be observed with our system.

Conclusions: We have presented a method to turn any commercial light microscope with monochromatic illumination into a PS QPI system. The proposed system provides accurate quantitative PS phase images in a new, simple, compact, and cost-effective format, thanks to the low cost (a few hundred dollars) involved in implementing this simple architecture, enabling the use of this QPI technique accessible to most laboratories with standard light microscopes.

Significance: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification.

Aim: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes.

Approach: The methodology was demonstrated by studying epithelial–mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images.

Results: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes.

Conclusions: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.

Significance: Functional near-infrared spectroscopy (fNIRS) is a technique for detecting regional hemodynamic responses associated with neural activation in the cerebral cortex. The absorption changes due to hemodynamic changes in the scalp cause considerable signal contamination in the fNIRS measurement. A method for extracting hemodynamic changes in the cerebral tissue is required for reliable fNIRS measurement.

Aim: To exclusively detect cerebral functional hemodynamic changes, we developed an fNIRS technique using reflectance modulation of the scalp surface.

Approach: The theoretical feasibility of the proposed method was proven by a simulation calculation of light propagation. Its practical feasibility was evaluated by a phantom experiment and brain activation simulation mimicking human fNIRS experiments.

Results: The simulation calculation revealed that the partial path length of the scalp was changed by reflectance modulation of the scalp surface. The influence of absorption change in the superficial layer was successfully reduced by the proposed method, using only measurement data, in the phantom experiment. The proposed method was applicable to human experiments of standard designs, achieving statistical significance within an acceptable experimental time-frame.

Conclusions: Removal of the scalp hemodynamic effect by the proposed technique will increase the quality of fNIRS data, particularly in measurements in neonates and infants that typically would require a dense optode arrangement.

Significance: A noninvasive method based on surface-enhanced Raman spectroscopy (SERS) of tears was proposed as a support for diagnosing neurodegenerative pathologies, including different forms of dementia and Alzheimer’s disease (AD). In this field, timely and reliable discrimination and diagnosis are critical aspects for choosing a valid medical therapy, and new methods are highly required.

Aim: The aim is to evince spectral differences in SERS response of human tears from AD affected, mild cognitive impaired (MCI), and healthy control (Ctr) subjects.

Approach: Human tears were characterized by SERS coupled with multivariate data analysis. Thirty-one informed subjects (Ctr, MCI, and AD) were considered.

Results: Average SERS spectra from Ctr, MCI, and AD subjects evidenced differences related to lactoferrin and lysozyme protein components. Quantitative changes were also observed by determining the intensity ratio between selected bands. We also constructed a classification model that discriminated among AD, MCI, and Ctr subjects. The model was built using the scores obtained by performing principal component analysis on specific spectral regions (i-PCA).

Conclusions: The results are very encouraging with interesting perspectives for medical applications as support of clinical diagnosis and discrimination of AD from other forms of dementia.