Spectral effects and enhancement quantification in healthy human saliva with surface-enhanced Raman spectroscopy using silver nanopillar substrates.

OBJECTIVES: Raman spectroscopy as a diagnostic tool for biofluid applications is limited by low inelastic scattering contributions compared to the fluorescence background from biomolecules. Surface-enhanced Raman spectroscopy (SERS) can increase Raman scattering signals, thereby offering the potential to reduce imaging times. We aimed to evaluate the enhancement related to the plasmonic effect and quantify the improvements in terms of spectral quality associated with SERS measurements in human saliva. METHODS: Dried human saliva was characterized using spontaneous Raman spectroscopy and SERS. A fabrication protocol was implemented leading to the production of silver (Ag) nanopillar substrates by glancing angle deposition. Two different imaging systems were used to interrogate saliva from 161 healthy donors: a custom single-point macroscopic system and a Raman micro-spectroscopy instrument. Quantitative metrics were established to compare spontaneous RS and SERS measurements: the Raman spectroscopy quality factor (QF), the photonic count rate (PR), the signal-to-background ratio (SBR). RESULTS: SERS measurements acquired with an excitation energy four times smaller than with spontaneous RS resulted in improved QF, PR values an order of magnitude larger and a SBR twice as large. The SERS enhancement reached 100×, depending on which Raman bands were considered. CONCLUSIONS: Single-point measurement of dried saliva with silver nanopillars substrates led to reproducible SERS measurements, paving the way to real-time tools of diagnosis in human biofluids.

Optimized selection of neodymium laser parameters for successful enlarged veins treatment.

Full clearance and no side effects method of treating enlarged veins was successfully accomplished by one laser session. This is the ambition and dream of both dermatologists and patients. Most vascularity treatment protocols had shown some unpleasant adverse effects. The purpose of the present work is to work out, in advance, the accurate dose of laser pulse duration and fluence in order to treat varicose veins in the face, arms and legs with no adverse effects. This mission required the calculation of the exact rise in temperature of the enlarged vein; prior to laser treatment. These pre-calculated temperature rise values were tested on 20 subjects in order to have the best clinical outcomes; using fundamental frequency (1064 nm) pulsed Nd: YAG laser. This work necessitated the use of pulse length (15-30 ms), spot size (3, 5 mm), fluence (110-190 J/cm2) and skin cooling temperature (3-18 °C). Cooling of the skin before and after the treatment was needed to guarantee ultimate impactful results without side effects.

Feasibility of Skin Water Content Imaging Using CMOS Sensors.

Pressure injuries (PI) result from pressure-induced damage to the skin and underlying tissues. Currently, Stage I PI are detected using visual skin assessments. However, this visual method is unable to detect skin color changes in persons with darkly pigmented skin, which results in a higher Stage II-IV PI incidence and PI-associated mortality in persons with a darker complexion. Thus, a more objective method of early-stage PI detection is of great importance. Optical spectroscopy is a promising modality for the noncontact diagnosis and monitoring of skin water content, capable of detecting edema and Stage I PI. The scope of the current study is to assess the feasibility of imaging the water content of the skin using Si-based sensors. We have considered two primary cases: the elevated bulk water content (edema) and localized water pool (e.g., blood vessels). These two cases were analyzed using analytical models. We found that detecting the watercontent contrast associated with edema in tissues is within the reach of Si-based sensors. However, although the effect is expected to be detectable even with consumer-grade cameras, with the current state of technologies, their use in real-world conditions faces numerous technical challenges, mainly due to the narrow dynamic range.

Improved Optical Tissue Model for Tissue Oximetry Imaging Applications.

INTRODUCTION: Chronic, non-healing wounds are a growing concern in healthcare delivery. Tissue oxygenation is recognised as critical to successful wound healing. However, the quality and quantity of the information extracted by hyperspectral imaging depend on the optical tissue model. This article aims to develop a simplified and computationally efficient approach comparable in quality with the two-layer model. METHODS: We have considered the epidermal layer as a 'thin-film' within the dermal layer. By considering the mismatched boundary and developing a four-flux model for light transport within the tissue, we have obtained a quasi two-layer model with a closed-form solution similar to the single-layer model. RESULTS: We have compared the developed model with the two-layer model (reference) and the single-layer model for the broad range of physiologically relevant parameters. The thickness of the epithelium: 50, 80, and 120 µm. Melanin concentration: 1, 2, 4, 8, 16, and 32%. Blood concentration: 0.2%, 1%, and 7%. Oxygen saturation: 60%, 80%, and 99%. Our initial results show that the accuracy of the proposed quasi two-layer model significantly (by a factor of 10) outperforms the single-layer model and is in close agreement with the two-layer model. CONCLUSIONS: The proposed quasi two-layer model significantly (by the factor of 10) outperforms the single-layer model and is closely aligned with the two-layer model.

An Evaluation Framework for Spectral Filter Array Cameras to Optimize Skin Diagnosis.

Comparing and selecting an adequate spectral filter array (SFA) camera is application-specific and usually requires extensive prior measurements. An evaluation framework for SFA cameras is proposed and three cameras are tested in the context of skin analysis. The proposed framework does not require application-specific measurements and spectral sensitivities together with the number of bands are the main focus. An optical model of skin is used to generate a specialized training set to improve spectral reconstruction. The quantitative comparison of the cameras is based on reconstruction of measured skin spectra, colorimetric accuracy, and oxygenation level estimation differences. Specific spectral sensitivity shapes influence the results directly and a 9-channel camera performed best regarding the spectral reconstruction metrics. Sensitivities at key wavelengths influence the performance of oxygenation level estimation the strongest. The proposed framework allows to compare spectral filter array cameras and can guide their application-specific development.

Characterization of a Time-Resolved Diffuse Optical Spectroscopy Prototype Using Low-Cost, Compact Single Photon Avalanche Detectors for Tissue Optics Applications.

Time-resolved diffuse optical spectroscopy (TR-DOS) is an increasingly used method to determine the optical properties of diffusive media, particularly for medical applications including functional brain, breast and muscle measurements. For medical imaging applications, important features of new generation TR-DOS systems are low-cost, small size and efficient inverse modeling. To address the issues of low-cost, compact size and high integration capabilities, we have developed free-running (FR) single-photon avalanche diodes (SPADs) using 130 nm silicon complementary metal-oxide-semiconductor (CMOS) technology and used it in a TR-DOS prototype. This prototype was validated using assessments from two known protocols for evaluating TR-DOS systems for tissue optics applications. Following the basic instrumental performance protocol, our prototype had sub-nanosecond total instrument response function and low differential non-linearity of a few percent. Also, using light with optical power lower than the maximum permissible exposure for human skin, this prototype can acquire raw data in reflectance geometry for phantoms with optical properties similar to human tissues. Following the MEDPHOT protocol, the absolute values of the optical properties for several homogeneous phantoms were retrieved with good accuracy and linearity using a best-fitting model based on the Levenberg-Marquardt method. Overall, the results of this study show that our silicon CMOS-based SPAD detectors can be used to build a multichannel TR-DOS prototype. Also, real-time functional monitoring of human tissue such as muscles, breasts and newborn heads will be possible by integrating this detector with a time-to-digital converter (TDC).

Optical-Based Analysis of Soft Tissue Structures.

Fibrous structures are an integral and dynamic feature of soft biological tissues that are directly related to the tissues' condition and function. A greater understanding of mechanical tissue behavior can be gained through quantitative analyses of structure alone, as well as its integration into computational models of soft tissue function. Histology and other nonoptical techniques have traditionally dominated the field of tissue imaging, but they are limited by their invasiveness, inability to provide resolution on the micrometer scale, and dynamic information. Recent advances in optical modalities can provide higher resolution, less invasive imaging capabilities, and more quantitative measurements. Here we describe contemporary optical imaging techniques with respect to their suitability in the imaging of tissue structure, with a focus on characterization and implementation into subsequent modeling efforts. We outline the applications and limitations of each modality and discuss the overall shortcomings and future directions for optical imaging of soft tissue structure.

Lateral light transfer ensures efficient resource distribution in symbiont-bearing corals.

Coral tissue optics has received very little attention in the past, although the interaction between tissue and light is central to our basic understanding of coral physiology. Here we used fibre-optic and electrochemical microsensors along with variable chlorophyll fluorescence imaging to directly measure lateral light propagation within living coral tissues. Our results show that corals can transfer light laterally within their tissues to a distance of ~2 cm. Such light transport stimulates O2 evolution and photosystem II operating efficiency in areas >0.5-1 cm away from direct illumination. Light is scattered strongly in both coral tissue and skeleton, leading to photon trapping and lateral redistribution within the tissue. Lateral light transfer in coral tissue is a new mechanism by which light is redistributed over the coral colony and we argue that tissue optical properties are one of the key factors in explaining the high photosynthetic efficiency of corals.

Tutorial on methods for estimation of optical absorption and scattering properties of tissue.

Significance: The estimation of tissue optical properties using diffuse optics has found a range of applications in disease detection, therapy monitoring, and general health care. Biomarkers derived from the estimated optical absorption and scattering coefficients can reflect the underlying progression of many biological processes in tissues. Aim: Complex light-tissue interactions make it challenging to disentangle the absorption and scattering coefficients, so dedicated measurement systems are required. We aim to help readers understand the measurement principles and practical considerations needed when choosing between different estimation methods based on diffuse optics. Approach: The estimation methods can be categorized as: steady state, time domain, time frequency domain (FD), spatial domain, and spatial FD. The experimental measurements are coupled with models of light-tissue interactions, which enable inverse solutions for the absorption and scattering coefficients from the measured tissue reflectance and/or transmittance. Results: The estimation of tissue optical properties has been applied to characterize a variety of ex vivo and in vivo tissues, as well as tissue-mimicking phantoms. Choosing a specific estimation method for a certain application has to trade-off its advantages and limitations. Conclusion: Optical absorption and scattering property estimation is an increasingly important and accessible approach for medical diagnosis and health monitoring.

Can diffuse reflectance spectroscopy identify shuntodynia in pediatric hydrocephalus patients?

Significance: Shuntodynia is patient reported pain at the site of the implanted ventriculoperitoneal (VP) shunt. Pediatric hydrocephalus requiring shunt placement is a chronic and prevalent standard of care treatment and requires lifetime management. Shuntodynia is a subjective measure of shunt dysfunction. Quantitative, white-light tissue spectroscopy could be used to objectively identify this condition in the clinic. Aim: Pediatric subjects were recruited for optical sensing during routine clinical follow-up visits, post-VP shunt implantations. Acquired optical signals were translated into skin-hemodynamic signatures and were compared between subjects that reported shuntodynia versus those that did not. Approach: Diffuse reflectance spectroscopy (DRS) measurements were collected between 450 and 700 nm using a single-channel fiber-optical probe from (N=35) patients. Multiple reflectance spectra were obtained by the attending physician from regions both proximal and distal to the VP shunt sites and from a matched contralateral site for each subject. Acquired reflectance spectra were processed quantitatively into functional tissue optical endpoints. A two-way, repeated measures analysis of variance was used to assess whether and which of the optical variables were statistically separable, across subjects with shuntodynia versus those without. Results: Analyses indicated that intrapatient differences in vascular oxygen saturation measured between shunt sites relative to that obtained at the scar or contralateral sites was significantly lower in the pain group. We also find that the total hemoglobin concentrations at the shunt site were lowest relative to the other sites for subjects reporting pain. These findings suggest that shuntodynia pain arises in the scalp tissue around the implanted shunts and may be caused due to hypoxia and inflammation. Conclusions: Optically derived hemodynamic variables were statistically significantly different in subjects presenting with shuntodynia relative to those without. DRS could provide a viable mode in routine bedside monitoring of subjects with VP shunts for clinical management and assessment of shuntodynia.

Teaching and learning in biophotonics: Crossing the bridge between educators and students.

As a rapidly growing field, biophotonics demonstrates an increasingly higher demand for interdisciplinary professionals and requires the implementation of a structured approach to educational and outreach activities focused on appropriate curriculum, and teaching and learning for audiences with diverse technical backgrounds and learning styles. Our study shows the main findings upon applying this approach to biophotonics workshops delivered 2 consecutive years while updating and improving learning outcomes, teaching strategies, workshop content based on student and teacher feedback. We provided resources for a variety of lecture-based, experimental, computer simulation activities. Quality of subject matter, teaching, and overall learning was rated as "Very good" or "Good" by 88%, 76%, and 82% of students in average, respectively. Application of our teaching strategies and materials during short- and long-term workshops/courses could potentially increase the interest in pursuing careers in the biophotonics field and related areas, leading to standardized approaches in designing education and outreach events across centers.

Toward noncontact macroscopic imaging of multiple cancers using multi-spectral inelastic scattering detection.

Here we introduce a Raman spectroscopy approach combining multi-spectral imaging and a new fluorescence background subtraction technique to image individual Raman peaks in less than 5 seconds over a square field-of-view of 1-centimeter sides with 350 micrometers resolution. First, human data is presented supporting the feasibility of achieving cancer detection with high sensitivity and specificity - in brain, breast, lung, and ovarian/endometrium tissue - using no more than three biochemically interpretable biomarkers associated with the inelastic scattering signal from specific Raman peaks. Second, a proof-of-principle study in biological tissue is presented demonstrating the feasibility of detecting a single Raman band - here the CH2/CH3 deformation bands from proteins and lipids - using a conventional multi-spectral imaging system in combination with the new background removal method. This study paves the way for the development of a new Raman imaging technique that is rapid, label-free, and wide field.

Macroscopic inelastic scattering imaging using a hyperspectral line-scanning system identifies invasive breast cancer in lumpectomy and mastectomy specimens.

Significance: Of patients with early-stage breast cancer, 60% to 75% undergo breast-conserving surgery. Of those, 20% or more need a second surgery because of an incomplete tumor resection only discovered days after surgery. An intraoperative imaging technology allowing cancer detection on the margins of breast specimens could reduce re-excision procedure rates and improve patient survival. Aim: We aimed to develop an experimental protocol using hyperspectral line-scanning Raman spectroscopy to image fresh breast specimens from cancer patients. Our objective was to determine whether macroscopic specimen images could be produced to distinguish invasive breast cancer from normal tissue structures. Approach: A hyperspectral inelastic scattering imaging instrument was used to interrogate eight specimens from six patients undergoing breast cancer surgery. Machine learning models trained with a different system to distinguish cancer from normal breast structures were used to produce tissue maps with a field-of-view of 1 cm 2 classifying each pixel as either cancer, adipose, or other normal tissues. The predictive model results were compared with spatially correlated histology maps of the specimens. Results: A total of eight specimens from six patients were imaged. Four of the hyperspectral images were associated with specimens containing cancer cells that were correctly identified by the new ex vivo pathology technique. The images associated with the remaining four specimens had no histologically detectable cancer cells, and this was also correctly predicted by the instrument. Conclusions: We showed the potential of hyperspectral Raman imaging as an intraoperative breast cancer margin assessment technique that could help surgeons improve cosmesis and reduce the number of repeat procedures in breast cancer surgery.

Surface-Enhanced Spatially Offset Raman Spectroscopy in Tissue.

One aim of personalized medicine is to use continuous or on-demand monitoring of metabolites to adjust prescription dosages in real time. Surface-enhanced spatially offset Raman spectroscopy (SESORS) is an optical technique capable of detecting surface-enhanced Raman spectroscopy (SERS)-active targets under a barrier, which may enable frequent metabolite monitoring. Here we investigate how the intensity of the signal from SERS-active material varies spatially through tissue, both experimentally and in a computational model. Implant-sized, SERS-active hydrogel was placed under different thicknesses of contiguous tissue. Emission spectra were collected at the air-tissue boundary over a range of offsets from the excitation site. New features were added to the Monte Carlo light-tissue interaction model to modify the optical properties after inelastic scattering and to calculate the distribution of photons as they exit the model. The Raman signals were detectable through all barrier thicknesses, with strongest emission for the case of 0 mm offset between the excitation and detector. A steep decline in the signal intensities occurred for offsets greater than 2 mm. These results did not match published SORS work (where targets were much larger than an implant). However, the model and experimental results agree in showing the greatest intensities at 0 mm offset and a steep gradient in the intensities with increasing offset. Also, the model showed an increase in the number of photons when the new, longer wavelengths were used following the Stokes shift for scattering and the graphical display of the exiting photons was helpful in the determination and confirmation of the optimal offset.

Modeling Light Scattering in Tissue as Continuous Random Media Using a Versatile Refractive Index Correlation Function.

Optical interactions with biological tissue provide powerful tools for study, diagnosis, and treatment of disease. When optical methods are used in applications involving tissue, scattering of light is an important phenomenon. In imaging modalities, scattering provides contrast, but also limits imaging depth, so models help optimize an imaging technique. Scattering can also be used to collect information about the tissue itself providing diagnostic value. Therapies involving focused beams require scattering models to assess dose distribution. In all cases, models of light scattering in tissue are crucial to correctly interpreting the measured signal. Here, we review a versatile model of light scattering that uses the Whittle-Matérn correlation family to describe the refractive index correlation function Bn (rd ). In weakly scattering media such as tissue, Bn (rd ) determines the shape of the power spectral density from which all other scattering characteristics are derived. This model encompasses many forms such as mass fractal and the Henyey-Greenstein function as special cases. We discuss normalization and calculation of optical properties including the scattering coefficient and anisotropy factor. Experimental methods using the model are also described to quantify tissue properties that depend on length scales of only a few tens of nanometers.

Lasers and laser-like devices: part one.

Lasers have been used in dermatology for nearly 50 years. Through their selective targeting of skin chromophores they have become the preferred treatment for many skin conditions, including vascular malformations, photorejuvenation and acne scars. The technology and design of lasers continue to evolve, allowing greater control of laser parameters and resulting in increased safety and efficacy for patients. Innovations have allowed the range of conditions and the skin types amenable to treatment, in both general and cosmetic dermatology, to expand over the last decade. Integrated skin cooling and laser beam fractionation, for example, have improved safety, patient tolerance and decreased downtime. Furthermore, the availability and affordability of quality devices continues to increase, allowing clinicians not only to access laser therapies more readily but also to develop their personal experience in this field. As a result, most Australian dermatologists now have access to laser therapies, either in their own practice or within referable proximity, and practical knowledge of these technologies is increasingly required and expected by patients. Non-laser energy devices utilising intense pulsed light, plasma, radiofrequency, ultrasound and cryolipolysis contribute to the modern laser practitioners' armamentarium and will also be discussed.

Hemodynamic alterations response to Chinese acupuncture therapy monitored by a custom near-infrared spectroscopy probe with an open hole.

Traditional acupuncture, a popular traditional Chinese medicine (TCM) technique, has demonstrated potential in relieving various ailments' symptoms. However, its black-box feedback loop model has limited proper evaluation and use by unskilled practitioners. This study aimed to analyze hemodynamic signals around acupoints during acupuncture to identify significant parameter changes. We designed hollow near-infrared spectroscopy (NIRS) probes for real-time measurements during acupuncture at acupoints, monitoring oxy-hemoglobin (HbO), deoxy-hemoglobin (Hb), and blood volume (HbT) changes. Acupuncture was performed on the "Xuehai" acupoint in 17 healthy subjects, with NIRS measuring hemodynamic alterations. Results showed significant and consistent increases in Hb and HbT around the acupoint, returning to baseline values after needle removal. One case of fainting revealed a significant Hb increase and HbO decrease. Acupuncture may induce tissue vasodilation and enhance oxygen consumption. This research provides a potential explanation for acupuncture's mechanism and emphasizes NIRS's potential in TCM.

Open-sourced Raman spectroscopy data processing package implementing a baseline removal algorithm validated from multiple datasets acquired in human tissue and biofluids.

Significance: Standardized data processing approaches are required in the field of bio-Raman spectroscopy to ensure information associated with spectral data acquired by different research groups, and with different systems, can be compared on an equal footing. Aim: An open-sourced data processing software package was developed, implementing algorithms associated with all steps required to isolate the inelastic scattering component from signals acquired using Raman spectroscopy devices. The package includes a novel morphological baseline removal technique (BubbleFill) that provides increased adaptability to complex baseline shapes compared to current gold standard techniques. Also incorporated in the package is a versatile tool simulating spectroscopic data with varying levels of Raman signal-to-background ratios, baselines with different morphologies, and varying levels of stochastic noise. Results: Application of the BubbleFill technique to simulated data demonstrated superior baseline removal performance compared to standard algorithms, including iModPoly and MorphBR. The data processing workflow of the open-sourced package was validated in four independent in-human datasets, demonstrating it leads to inter-systems data compatibility. Conclusions: A new open-sourced spectroscopic data pre-processing package was validated on simulated and real-world in-human data and is now available to researchers and clinicians for the development of new clinical applications using Raman spectroscopy.

A Comparison of Spectroscopy and Imaging Techniques Utilizing Spectrally Resolved Diffusely Reflected Light for Intraoperative Margin Assessment in Breast-Conserving Surgery: A Systematic Review and Meta-Analysis.

Up to 19% of patients require re-excision surgery due to positive margins in breast-conserving surgery (BCS). Intraoperative margin assessment tools (IMAs) that incorporate tissue optical measurements could help reduce re-excision rates. This review focuses on methods that use and assess spectrally resolved diffusely reflected light for breast cancer detection in the intraoperative setting. Following PROSPERO registration (CRD42022356216), an electronic search was performed. The modalities searched for were diffuse reflectance spectroscopy (DRS), multispectral imaging (MSI), hyperspectral imaging (HSI), and spatial frequency domain imaging (SFDI). The inclusion criteria encompassed studies of human in vivo or ex vivo breast tissues, which presented data on accuracy. The exclusion criteria were contrast use, frozen samples, and other imaging adjuncts. 19 studies were selected following PRISMA guidelines. Studies were divided into point-based (spectroscopy) or whole field-of-view (imaging) techniques. A fixed-or random-effects model analysis generated pooled sensitivity/specificity for the different modalities, following heterogeneity calculations using the Q statistic. Overall, imaging-based techniques had better pooled sensitivity/specificity (0.90 (CI 0.76-1.03)/0.92 (CI 0.78-1.06)) compared with probe-based techniques (0.84 (CI 0.78-0.89)/0.85 (CI 0.79-0.91)). The use of spectrally resolved diffusely reflected light is a rapid, non-contact technique that confers accuracy in discriminating between normal and malignant breast tissue, and it constitutes a potential IMA tool.

Subsecond lung cancer detection within a heterogeneous background of normal and benign tissue using single-point Raman spectroscopy.

Significance: Lung cancer is the most frequently diagnosed cancer overall and the deadliest cancer in North America. Early diagnosis through current bronchoscopy techniques is limited by poor diagnostic yield and low specificity, especially for lesions located in peripheral pulmonary locations. Even with the emergence of robotic-assisted platforms, bronchoscopy diagnostic yields remain below 80%. Aim: The aim of this study was to determine whether in situ single-point fingerprint (800 to 1700 cm-1) Raman spectroscopy coupled with machine learning could detect lung cancer within an otherwise heterogenous background composed of normal tissue and tissue associated with benign conditions, including emphysema and bronchiolitis. Approach: A Raman spectroscopy probe was used to measure the spectral fingerprint of normal, benign, and cancer lung tissue in 10 patients. Each interrogated specimen was characterized by histology to determine cancer type, i.e., small cell carcinoma or non-small cell carcinoma (adenocarcinoma and squamous cell carcinoma). Biomolecular information was extracted from the fingerprint spectra to identify biomolecular features that can be used for cancer detection. Results: Supervised machine learning models were trained using leave-one-patient-out cross-validation, showing lung cancer could be detected with a sensitivity of 94% and a specificity of 80%. Conclusions: This proof of concept demonstrates fingerprint Raman spectroscopy is a promising tool for the detection of lung cancer during diagnostic procedures and can capture biomolecular changes associated with the presence of cancer among a complex heterogeneous background within less than 1 s.