Self-supervised denoising of Nyquist-sampled volumetric images via deep learning.

Purpose: Deep learning has demonstrated excellent performance enhancing noisy or degraded biomedical images. However, many of these models require access to a noise-free version of the images to provide supervision during training, which limits their utility. Here, we develop an algorithm (noise2Nyquist) that leverages the fact that Nyquist sampling provides guarantees about the maximum difference between adjacent slices in a volumetric image, which allows denoising to be performed without access to clean images. We aim to show that our method is more broadly applicable and more effective than other self-supervised denoising algorithms on real biomedical images, and provides comparable performance to algorithms that need clean images during training. Approach: We first provide a theoretical analysis of noise2Nyquist and an upper bound for denoising error based on sampling rate. We go on to demonstrate its effectiveness in denoising in a simulated example as well as real fluorescence confocal microscopy, computed tomography, and optical coherence tomography images. Results: We find that our method has better denoising performance than existing self-supervised methods and is applicable to datasets where clean versions are not available. Our method resulted in peak signal to noise ratio (PSNR) within 1 dB and structural similarity (SSIM) index within 0.02 of supervised methods. On medical images, it outperforms existing self-supervised methods by an average of 3 dB in PSNR and 0.1 in SSIM. Conclusion: noise2Nyquist can be used to denoise any volumetric dataset sampled at at least the Nyquist rate making it useful for a wide variety of existing datasets.

Optical coherence tomography holds promise to transform the diagnostic anatomic pathology gross evaluation process.

SIGNIFICANCE: Real-time histology can close a variety of gaps in tissue diagnostics. Currently, gross pathology analysis of excised tissue is dependent upon visual inspection and palpation to identify regions of interest for histopathological processing. Such analysis is limited by the variable correlation between macroscopic and microscopic findings. The current standard of care is costly, burdensome, and inefficient. AIM: We are the first to address this gap by introducing optical coherence tomography (OCT) to be integrated in real-time during the pathology grossing process. APPROACH: This is achieved by our high-resolution, ultrahigh-speed, large field-of-view OCT device designed for this clinical application. RESULTS: We demonstrate the feasibility of imaging tissue sections from multiple human organs (breast, prostate, lung, and pancreas) in a clinical gross pathology setting without interrupting standard workflows. CONCLUSIONS: OCT-based real-time histology evaluation holds promise for addressing a gap that has been present for >100 years.

Hemodynamics of the sternocleidomastoid measured with frequency domain near-infrared spectroscopy towards non-invasive monitoring during mechanical ventilation.

Mechanical ventilation (MV) is used to assist spontaneous breathing in critically ill patients in the intensive care unit (ICU). MV is a cornerstone of critical care medicine but it is now known that inspiratory muscle dysfunction due to injury, disuse, and/or atrophy during MV plays a major role in outcomes for these patients. For example, prolonged MV is strongly correlated with dysfunction of the sternocleidomastoid (SCM), an accessory inspiratory muscle that has been linked to weaning failure from MV. Hemodynamic monitoring of the SCM may provide an important non-invasive and real-time means to monitor MV. In this work, we first conducted multi-layer Monte Carlo simulations to confirm the ability of near infrared light to detect changes in the oxygenation of the SCM over wide ranges of skin tones and adipose layer thicknesses. We then optimized a custom digital frequency domain near-infrared spectroscopy (FD-NIRS) system for continuous 10 Hz measurements of the SCM at 730 nm and 850 nm. A healthy volunteer study was conducted (N=10); subjects performed sets of isometric neck flexions of the SCM. Substantial changes in oxyhemoglobin + oxymyoglobin (oxy[Hb + Mb]), deoxyhemoglobin + deoxymyoglobin (deoxy[Hb + Mb]), and total hemoglobin + myoglobin (total[Hb + Mb]) were observed during sustained and intermittent isometric flexions. There were notable sex differences observed in the magnitude of hemodynamic changes (∼2x larger changes in males for oxy[Hb + Mb] and deoxy[Hb + Mb]). The magnitude of hemodynamic changes when taking into account µs' changes during flexions was ∼ 2-2.5x larger as compared to assuming constant scattering (CS), which is a common assumption used for continuous wave (CW) NIRS methods. This study suggests that FD-NIRS provides improved accuracy for hemodynamic monitoring of the SCM compared to CW-NIRS, and that FD-NIRS may provide value for SCM monitoring during MV.

Real-Time Handheld Probe Tracking and Image Formation Using Digital Frequency-Domain Diffuse Optical Spectroscopy.

OBJECTIVE: Frequency-domain diffuse optical spectroscopic imaging (FD-DOS) is a non-invasive method for measuring absolute concentrations of tissue chromophores such as oxy- and deoxy-hemoglobin in vivo. The utility of FD-DOS for clinical applications such as monitoring chemotherapy response in breast cancer has previously been demonstrated, but challenges for further clinical translation, such as slow acquisition speed and lack of user feedback, remain. Here, we propose a new high speed FD-DOS instrument that allows users to freely acquire measurements over the tissue surface, and is capable of rapidly imaging large volumes of tissue. METHODS: We utilize 3D monocular probe tracking combined with custom digital FD-DOS hardware and a high-speed data processing pipeline for the instrument. Results are displayed during scanning over the surface of the sample using a probabilistic Monte Carlo light propagation model. RESULTS: We show this instrument can measure absorption and scattering coefficients with an error of 7% and 1% respectively, with 0.7 mm positional accuracy. We demonstrate the equivalence of our visualization methodology with a standard interpolation approach, and demonstrate two proof-of-concept in vivo results showing superficial vasculature in the human forearm and surface contrast in a healthy human breast. CONCLUSION: Our new FD-DOS system is able to compute chromophore concentrations in real-time (1.5 Hz) in vivo. SIGNIFICANCE: This method has the potential to improve the quality of FD-DOS image scans while reducing measurement times for a variety of clinical applications.

Modulation frequency selection and efficient look-up table inversion for frequency domain diffuse optical spectroscopy.

SIGNIFICANCE: Frequency domain diffuse optical spectroscopy (FD-DOS) uses intensity modulated light to measure the absorption and reduced scattering coefficients of turbid media such as biological tissue. Some FD-DOS instruments utilize a single modulation frequency, whereas others use hundreds of frequencies. The effect of modulation frequency choice and measurement bandwidth on optical property (OP) extraction accuracy has not yet been fully characterized. AIM: We aim to assess the effect of modulation frequency selection on OP extraction error and develop a high-speed look-up table (LUT) approach for OP estimation. APPROACH: We first used noise-free simulations of light transport in homogeneous media to determine optimized iterative inversion model parameters and developed a new multi-frequency LUT method to increase the speed of inversion. We then used experimentally derived noise models for two FD-DOS instruments to generate realistic simulated data for a broad range of OPs and modulation frequencies to test OP extraction accuracy. RESULTS: We found that repeated measurements at a single low-frequency (110 MHz) yielded essentially identical OP errors as a broadband frequency sweep (35 evenly spaced frequencies between 50 and 253 MHz) for these noise models. The inclusion of modulation frequencies >300 MHz diminished overall performance for one of the instruments. Additionally, we developed a LUT inversion algorithm capable of increasing inversion speeds by up to 6 × , with 1000 inversions / s and ∼1 % error when a single modulation frequency was used. CONCLUSION: These results suggest that simpler single-frequency systems are likely sufficient for many applications and pave the way for a new generation of simpler digital FD-DOS systems capable of rapid, large-volume measurements with real-time feedback.

Dual-DMD hyperspectral spatial frequency domain imaging (SFDI) using dispersed broadband illumination with a demonstration of blood stain spectral monitoring.

Spatial frequency domain imaging (SFDI) is a widefield diffuse optical measurement technique capable of generating 2D maps of sub-surface absorption and scattering in biological tissue. We developed a new hyperspectral SFDI instrument capable of collecting images at wavelengths from the visible to the near infrared. The system utilizes a custom-built monochromator with a digital micromirror device (DMD) that can dynamically select illumination wavelength bands from a broadband quartz tungsten halogen lamp, and a second DMD to provide spatially modulated sample illumination. The system is capable of imaging 10 wavelength bands in approximately 25 seconds. The spectral resolution can be varied from 12 to 30 nm by tuning the input slit width and the output DMD column width. We compared the optical property extraction accuracy between the new device and a commercial SFDI system and found an average error of 23% in absorption and 6% in scattering. The system was highly stable, with less than 5% variation in absorption and less than 0.2% variation in scattering across all wavelengths over two hours. The system was used to monitor hyperspectral changes in the optical absorption and reduced scattering spectra of blood exposed to air over 24 hours. This served as a general demonstration of the utility of this system, and points to a potential application for blood stain age estimation. We noted significant changes in both absorption and reduced scattering spectra over multiple discrete stages of aging. To our knowledge, these are the first measurement of changes in scattering of blood stains. This hyperspectral SFDI system holds promise for a multitude of applications in quantitative tissue and diffuse sample imaging.

Auricular acupuncture for the treatment of nonepileptic seizures: A pilot study.

OBJECTIVE: The aim of this pilot study was to examine the safety and tolerability of an eight-week treatment of twice weekly auricular acupuncture as a therapeutic intervention for the treatment of psychogenic nonepileptic seizures (PNES). The secondary aim was to determine if auricular acupuncture was effective at reducing PNES event frequency. Psychogenic nonepileptic seizures are as common as multiple sclerosis and are highly resistant to intervention. Unfortunately, there is no standard of care therapy, although mental health treatment referrals are commonly made. Acupuncture is felt to be a safe and cost-effective treatment for many diseases and symptoms including mental health disorders such as posttraumatic stress disorder (PTSD), anxiety, and conversion disorders. We propose to examine auricular acupuncture as a treatment for PNES. METHODS: A single-center, 16-week trial of auricular acupuncture was designed: a four-week baseline period, followed by an eight-week active treatment period during which subjects received twice weekly auricular acupuncture, then followed by a four-week washout period. Tolerability was defined as completion of 12 of 16 treatments. Successful compliance with seizure diary was defined as >80%, or 100 of 120 days completed. Safety profile was established and seizure count per week was also calculated. RESULTS: A total of 29 subjects enrolled from a consecutive sample of 73 PNES diagnoses discharging from our epilepsy unit. Twenty-two subjects entered the study with 91% completing more than 12 of 16 treatments, 91% meeting the endpoint of completing >80% of diary entries. Adverse events were mild, and only two subjects were withdrawn due to increased PNES frequency. Although short-term in design, using one-way repeated measures analysis of variance (ANOVA), we observed a statistically significant reduction in seizure count from baseline at treatment month one, treatment month two, and at the washout period (p = 0.0083). Eleven of 12 subjects (52.3%) who received at least one treatment recorded a 50% frequency reduction. Three of 21 subjects (14%) were PNES-free at the end of the study. SIGNIFICANCE: Auricular acupuncture is well-tolerated in a population with PNES and exhibits similar event frequency reductions as cognitive behavioral therapy (CBT). We believe that this is the first literature report examining auricular acupuncture for the treatment of PNES.

OpenSFDI: an open-source guide for constructing a spatial frequency domain imaging system.

Significance: Spatial frequency domain imaging (SFDI) is a diffuse optical measurement technique that can quantify tissue optical absorption (µa) and reduced scattering (µs') on a pixel-by-pixel basis. Measurements of µa at different wavelengths enable the extraction of molar concentrations of tissue chromophores over a wide field, providing a noncontact and label-free means to assess tissue viability, oxygenation, microarchitecture, and molecular content. We present here openSFDI: an open-source guide for building a low-cost, small-footprint, three-wavelength SFDI system capable of quantifying µa and µs' as well as oxyhemoglobin and deoxyhemoglobin concentrations in biological tissue. The companion website provides a complete parts list along with detailed instructions for assembling the openSFDI system.

Aim: We describe the design of openSFDI and report on the accuracy and precision of optical property extractions for three different systems fabricated according to the instructions on the openSFDI website.

Approach: Accuracy was assessed by measuring nine tissue-simulating optical phantoms with a physiologically relevant range of µa and µs' with the openSFDI systems and a commercial SFDI device. Precision was assessed by repeatedly measuring the same phantom over 1 h.

Results: The openSFDI systems had an error of 0 ± 6 % in µa and -2 ± 3 % in µs', compared to a commercial SFDI system. Bland-Altman analysis revealed the limits of agreement between the two systems to be ± 0.004 mm - 1 for µa and -0.06 to 0.1 mm - 1 for µs'. The openSFDI system had low drift with an average standard deviation of 0.0007 mm - 1 and 0.05 mm - 1 in µa and µs', respectively.

Conclusion: The openSFDI provides a customizable hardware platform for research groups seeking to utilize SFDI for quantitative diffuse optical imaging.

Distinguishing Tumor from Associated Fibrosis to Increase Diagnostic Biopsy Yield with Polarization-Sensitive Optical Coherence Tomography.

PURPOSE: With recent advancements in personalized medicine, biopsies must contain sufficient tumor for histologic diagnosis and molecular testing. However, inadvertent biopsy of tumor-associated fibrosis compromises tumor yield, resulting in delayed diagnoses and/or repeat procedures when additional tumor is needed. The ability to differentiate tumor from fibrosis intraprocedurally during biopsy could significantly increase tumor yield. Polarization-sensitive optical coherence tomography (PS-OCT) is an imaging modality that is endoscope- and/or needle-compatible, and provides large volumetric views of tissue microstructure with high resolution (∼10 µm) while simultaneously measuring birefringence of organized tissues such as collagen. We aim to determine whether PS-OCT can accurately detect and distinguish tumor-associated fibrosis from tumor. EXPERIMENTAL DESIGN: PS-OCT was obtained ex vivo in 64 lung nodule samples. PS-OCT birefringence was measured and correlated to collagen content in precisely matched histology, quantified on picrosirius red (PSR) staining. RESULTS: There was a strong positive correlation between PS-OCT measurement of birefringent fibrosis and total collagen content by PSR (r = 0.793; P < 0.001). In addition, PS-OCT was able to accurately classify tumor regions with >20% fibrosis from those with low fibrosis (≤20%) that would likely yield higher tumor content (P < 0.0001). CONCLUSIONS: PS-OCT enables accurate fibrosis detection and can distinguish tumor regions with low fibrosis. PS-OCT has significant potential for clinical impact, as the ability to differentiate tumor from fibrosis could be used to guide intraprocedural tissue sampling in vivo, or for rapid biopsy adequacy assessment ex vivo, to increase diagnostic tumor yield essential for patient care and research.

Quantitative assessment of airway remodelling and response to allergen in asthma.

BACKGROUND AND OBJECTIVE: In vivo evaluation of the microstructural differences between asthmatic and non-asthmatic airways and their functional consequences is relevant to understanding and, potentially, treating asthma. In this study, we use endobronchial optical coherence tomography to investigate how allergic airways with asthma differ from allergic non-asthmatic airways in baseline microstructure and in response to allergen challenge. METHODS: A total of 45 subjects completed the study, including 20 allergic, mildly asthmatic individuals, 22 non-asthmatic allergic controls and 3 healthy controls. A 3-cm airway segment in the right middle and right upper lobe were imaged in each subject immediately before and 24 h following segmental allergen challenge to the right middle lobe. Relationships between optical airway measurements (epithelial and mucosal thicknesses, mucosal buckling and mucus) and airway obstruction (FEV1 /FVC (forced expiratory volume in 1 s/forced vital capacity) and FEV1 % (FEV1 as a percentage of predictive value)) were investigated. RESULTS: Significant increases at baseline and in response to allergen were observed for all four of our imaging metrics in the asthmatic airways compared to the non-asthmatic airways. Epithelial thickness and mucosal buckling exhibited a significant relationship to FEV1 /FVC in the asthmatic group. CONCLUSION: Simultaneous assessments of airway microstructure, buckling and mucus revealed both structural and functional differences between the mildly asthmatic and control groups, with airway buckling seeming to be the most relevant factor. The results of this study demonstrate that a comprehensive, microstructural approach to assessing the airways may be important in future asthma studies as well as in the monitoring and treatment of asthma.

Multi-distance diffuse optical spectroscopy with a single optode via hypotrochoidal scanning.

Frequency-domain diffuse optical spectroscopy (FD-DOS) is an established technique capable of determining optical properties and chromophore concentrations in biological tissue. Most FD-DOS systems use either manually positioned, handheld probes or complex arrays of source and detector fibers to acquire data from many tissue locations, allowing for the generation of 2D or 3D maps of tissue. Here, we present a new method to rapidly acquire a wide range of source-detector (SD) separations by mechanically scanning a single SD pair. The source and detector fibers are mounted on a scan head that traces a hypotrochoidal pattern over the sample that, when coupled with a high-speed FD-DOS system, enables the rapid collection of dozens of SD separations for depth-resolved imaging. We demonstrate that this system has an average error of 4±2.6% in absorption and 2±1.8% in scattering across all SD separations. Additionally, by linearly translating the device, the size and location of an absorbing inhomogeneity can be determined through the generation of B-scan images in a manner conceptually analogous to ultrasound imaging. This work demonstrates the potential of single optode diffuse optical scanning for depth resolved visualization of heterogeneous biological tissues at near real-time rates.

High-speed spatial frequency domain imaging with temporally modulated light.

Spatial frequency domain imaging (SFDI) is a wide-field diffuse optical technique used to obtain optical properties and chromophore concentrations in highly scattering media, such as biological tissue. Here, we present a method for rapidly acquiring multispectral SFDI data by modulating each illumination wavelength at a different temporal frequency. In the remitted signal, each wavelength is temporally demodulated and processed using conventional SFDI techniques. We demonstrate a proof-of-concept system capable of acquiring wide-field maps (2048×1536 pixels, 8.5×6.4 cm) of optical properties at three wavelengths in under 2.5 s. Data acquired by this method show a good agreement with a commercial SFDI imaging system (with an average error of 13% in absorption and 8% in scattering). Additionally, we show that this strategy is insensitive to ambient lighting conditions, making it more practical for clinical translation. In the future, this technique could be expanded to tens or hundreds of wavelengths without increasing acquisition time.

Photo-induced structural modification of silk gels containing azobenzene side groups.

Azobenzene modification of Bombyx mori silkworm silk creates a photo-responsive 'azosilk' biomaterial, allowing for 3D laser patterning. Written regions fluoresce, and become fluid-filled raised 'micro-blisters' with a 10-fold photo-softening effect of the modulus. Patterning is facile and versatile, with potential applications as soft tunable materials for dynamic cell guidance and microfluidics.

Directed assembly of bio-inspired hierarchical materials with controlled nanofibrillar architectures.

In natural systems, directed self-assembly of structural proteins produces complex, hierarchical materials that exhibit a unique combination of mechanical, chemical and transport properties. This controlled process covers dimensions ranging from the nano- to the macroscale. Such materials are desirable to synthesize integrated and adaptive materials and systems. We describe a bio-inspired process to generate hierarchically defined structures with multiscale morphology by using regenerated silk fibroin. The combination of protein self-assembly and microscale mechanical constraints is used to form oriented, porous nanofibrillar networks within predesigned macroscopic structures. This approach allows us to predefine the mechanical and physical properties of these materials, achieved by the definition of gradients in nano- to macroscale order. We fabricate centimetre-scale material geometries including anchors, cables, lattices and webs, as well as functional materials with structure-dependent strength and anisotropic thermal transport. Finally, multiple three-dimensional geometries and doped nanofibrillar constructs are presented to illustrate the facile integration of synthetic and natural additives to form functional, interactive, hierarchical networks.

Evaluation of Silk Inverse Opals for "Smart" Tissue Culture.

Visually tracking the subtle aspects of biological systems in real time during tissue culture remains challenging. Herein, we demonstrate the use of bioactive, cytocompatible, and biodegradable inverse opals from silk as a multifunctional substrate to transduce both the optical information and cells during tissue culture. We show that these substrates can visually track substrate degradation in various proteases during tissue digestion and protein deposition during the growth of mesenchymal stem cells. Uniquely, these substrates can be integrated in multiple steps of tissue culture for simple-to-use, visual, and quantitative detectors of bioactivity. These substrates can also be doped, demonstrated here with gold nanoparticles, to allow additional control of cell functions.

Bio-functionalized silk hydrogel microfluidic systems.

Bio-functionalized microfluidic systems were developed based on a silk protein hydrogel elastomeric materials. A facile multilayer fabrication method using gelatin sacrificial molding and layer-by-layer assembly was implemented to construct interconnected, three dimensional (3D) microchannel networks in silk hydrogels at 100 µm minimum feature resolution. Mechanically activated valves were implemented to demonstrate pneumatic control of microflow. The silk hydrogel microfluidics exhibit controllable mechanical properties, long-term stability in various environmental conditions, tunable in vitro and in vivo degradability in addition to optical transparency, providing unique features for cell/tissue-related applications than conventional polydimethylsiloxane (PDMS) and existing hydrogel-based microfluidic options. As demonstrated in the work here, the all aqueous-based fabrication process at ambient conditions enabled the incorporation of active biological substances in the bulk phase of these new silk microfluidic systems during device fabrication, including enzymes and living cells, which are able to interact with the fluid flow in the microchannels. These silk hydrogel-based microfluidic systems offer new opportunities in engineering active diagnostic devices, tissues and organs that could be integrated in vivo, and for on-chip cell sensing systems.

Photocrosslinking of Silk Fibroin Using Riboflavin for Ocular Prostheses.

A novel method to photocrosslink silk fibroin protein is reported, using riboflavin (vitamin B2) as a photoinitiator and the mechanism of crosslinking is determined. Exposure of riboflavin-doped liquid silk solution to light results in the formation of a transparent, elastic hydrogel. Several applications for this new material are investigated including corneal reshaping to restore visual acuity and photolithography.

Dityrosine Cross-Linking in Designing Biomaterials.

The development of functional biomaterials for tissue engineering and medical applications has received increasing attention. While it has been known for decades that dityrosine bonds are a key component to many biopolymer materials in native tissues, only recently have these motifs been exploited in the development of new biomaterials. Here, we first review the importance of tyrosine-tyrosine chemical bonds in the assembly and mechanical properties of natural materials. Next, we discuss the chemistries available for cross-linking via tyrosine bonds and how these interactions have been applied to biomaterials. The goal of this Review is to highlight dityrosine bonding in biomaterial development, the reactions used to form them, and their utility in cross-linking native and chemically substituted phenolic side chains, as an underutilized tool in the de novo development of biomaterials.

Biocompatible silk step-index optical waveguides.

Biocompatible optical waveguides were constructed entirely of silk fibroin. A silk film (n=1.54) was encapsulated within a silk hydrogel (n=1.34) to form a robust and biocompatible waveguide. Such waveguides were made using only biologically and environmentally friendly materials without the use of harsh solvents. Light was coupled into the silk waveguides by direct incorporation of a glass optical fiber. These waveguides are extremely flexible, and strong enough to survive handling and manipulation. Cutback measurements showed propagation losses of approximately 2 dB/cm. The silk waveguides were found to be capable of guiding light through biological tissue.

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds.

Light-induced material phase transitions enable the formation of shapes and patterns from the nano- to the macroscale. From lithographic techniques that enable high-density silicon circuit integration, to laser cutting and welding, light-matter interactions are pervasive in everyday materials fabrication and transformation. These noncontact patterning techniques are ideally suited to reshape soft materials of biological relevance. We present here the use of relatively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and 3D multiscale patterns in soft silk protein hydrogels without exogenous or chemical cross-linkers. We find that high-resolution features can be generated within bulk hydrogels through nearly 1 cm of material, which is 1.5 orders of magnitude deeper than other biocompatible materials. Examples illustrating the materials, results, and the performance of the machined geometries in vitro and in vivo are presented to demonstrate the versatility of the approach.