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Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a "traffic light type μPAD", enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/greenyellow/yellow/yellowred/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H

Microfluidic paper-based analytical devices (microPADs) are emerging as simple-to-use, low-cost point-of-care testing platforms. Such devices are mostly fabricated at present by creating hydrophobic barriers using wax or photoresist patterning on porous paper sheets. Even though devices fabricated using these methods are used and tested with a wide variety of analytes, still they pose many serious practical limitations for low-cost automated mass fabrication for their widespread applicability. We present an affordable and simple two-step process - cut and heat (CH-microPADs) - for the selective fabrication of hydrophilic channels and reservoirs on a wide variety of porous media such as tissue/printing/filter paper and cloth types, such as cotton and polyester, by a lamination process. The technique presents many advantages as compared to existing commonly used methods. The devices possess excellent mechanical strength against bending, folding and twisting, making them virtually unbreakable. They are structurally flexible and show good chemical resistance to various solvents, acids and bases, presenting widespread applicability in areas such as clinical diagnostics, biological sensing applications, food processing, and the chemical industry. Fabricated paper media 96 well-plate CH-microPAD configurations were tested for cell culture applications using mice embryonic fibroblasts and detection of proteins and enzymes using ELISA. With a simple two-step process and minimal human intervention, the technique presents a promising step towards mass fabrication of inexpensive disposable diagnostic devices for both resource-limited and developed regions.

Environmental toxic pollutants and pathogens that enter the ecosystem are major global issues. Detection of these toxic chemicals/pollutants and the diagnosis of a disease is a first step in efficiently controlling their contamination and spread, respectively. Various analytical techniques are available to detect and determine toxic chemicals/pathogens, including liquid chromatography, HPLC, mass spectroscopy, and enzyme-linked immunosorbent assays. However, these sensing strategies have some drawbacks such as tedious sample pretreatment and preparation, the requirement for skilled technicians, and dependence on large laboratory-based instruments. Alternatively, biosensors, especially paper-based sensors, could be used extensively and are a cost-effective alternative to conventional laboratory testing. They can improve accessibility to testing to identify chemicals and pollutants, especially in developing countries. Due to its low cost, abundance, easy disposal (by incineration, for example) and biocompatible nature, paper is considered a versatile material for the development of environmentally friendly electrochemical/optical (bio) sensor devices. This review presents an overview of sensing platforms constructed from paper, pointing out the main merits and demerits of paper-based sensing systems, their fabrication techniques, and the different optical/electrochemical detection techniques that they exploit.

Aβ42 is one of the most extensively studied blood and Cerebrospinal fluid (CSF) biomarkers for the diagnosis of symptomatic and prodromal Alzheimer’s disease (AD). Because of the heterogeneity and transient nature of Aβ42 oligomers (Aβ42Os), the development of technologies for dynamically detecting changes in the blood or CSF levels of Aβ42 monomers (Aβ42Ms) and Aβ42Os is essential for the accurate diagnosis of AD. The currently commonly used Aβ42 ELISA test kits usually mis-detected the elevated Aβ42Os, leading to incomplete analysis and underestimation of soluble Aβ42, resulting in a comprised performance in AD diagnosis. Herein, we developed a dual-target lateral flow immunoassay (dLFI) using anti-Aβ42 monoclonal antibodies 1F12 and 2C6 for the rapid and point-of-care detection of Aβ42Ms and Aβ42Os in blood samples within 30 min for AD diagnosis. By naked eye observation, the visual detection limit of Aβ42Ms or/and Aβ42Os in dLFI was 154 pg/mL. The test results for dLFI were similar to those observed in the enzyme-linked immunosorbent assay (ELISA). Therefore, this paper-based dLFI provides a practical and rapid method for the on-site detection of two biomarkers in blood or CSF samples without the need for additional expertise or equipment. Graphical Abstract

This article reports a simple and inexpensive leak-proof paper pad with an initial selection of a paper substrate on the grounds of surface morphology and fluid absorption time. Herein, a drying method is used for glucose detection on a paper pad through colorimetric analysis, and the spot detection of glucose is analyzed by optimizing the HRP concentration and volume to obtain accurate results. The rapid colorimetric method for the detection of glucose on the paper pad was developed with a limit of detection (LOD) of 2.92 mmol L−1. Furthermore, the effects of the detection conditions were investigated and discussed comprehensively with the help of chemometric methods. Paper pads were developed for glucose detection with a range of 0.5–20 mM (apropos to the normal glucose level in the human body) and 0.1–0.5 M (to test the excessive intake of glucose). The developed concept has huge potential in the healthcare sector, and its extension could be envisioned to develop the reported paper pad as a point-of-care testing device for the initial screening of a variety of diseases.

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

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers. Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

Microfluidic paper-based analytical devices (μPADs) have been widely explored for point-of-care testing due to their simplicity, low cost, and portability. μPADs with multiple-step reactions usually require precise flow control, especially flow-delay. This paper reports the numerical, mathematical, and experimental studies of flow delay through wax valves surrounded by PDMS walls on paper microfluidics. The predried surfactant in the sample zone diffuses into the liquid sample which can therefore flow through the wax valves. The delay time is automatically regulated by the diffusion of the surfactant after sample loading. The numerical study suggested that both the elevated contact angle and the reduced porosity and pore size in the wax printed region could effectively prevent water but allow liquids with lower contact angles (e.g., surfactant solutions) to flow through. The PDMS walls fabricated using a low-cost liquid dispenser effectively prevented the leakage of surfactant solutions. By controlling the quantity, diffusion distance, and type of the surfactant predried on the chip, the system successfully achieved a delay time ranging from 1.6 to 20 minutes. A mathematical model involving the above parameters was developed based on Fick's second law to predict the delay time. Finally, the flow-delay systems were applied in sequential mixing and distance-based detection of either glucose or alcohol. Linear ranges of 1-100 mg dL-1 and 1-40 mg dL-1 were achieved for glucose and alcohol, respectively. The lower limit detection (LOD) of glucose and alcohol was 1 mg dL-1. The LOD of glucose was only 1/11 of that detected using μPADs without flow control, indicating the advantage of controlling fluid flow. The systematic findings in this study provide critical guidelines for the development and applications of wax valves in automatic flow delay for point-of-care testing. This journal is

Lab-on-paper, or microfluidic paper-based analytical devices (μPADs), use paper as a substrate material, and are patterned with a system of microchannels, reaction zones and sensing elements to perform analysis and detection. The sample transfer in such devices is performed by capillary action. As a result, external driving forces are not required, and hence the size and cost of the device are significantly reduced. Lab-on-paper devices have thus attracted significant attention for point-of-care medical diagnostic purposes in recent years, particularly in less-developed regions of the world lacking medical resources and infrastructures. This review discusses the major advances in lab-on-paper technology for blood analysis and diagnosis in the past five years. The review focuses particularly on the many clinical applications of lab-on-paper devices, including diabetes diagnosis, acute myocardial infarction (AMI) detection, kidney function diagnosis, liver function diagnosis, cholesterol and triglyceride (TG) analysis, sickle-cell disease (SCD) and phenylketonuria (PKU) analysis, virus analysis, C-reactive protein (CRP) analysis, blood ion analysis, cancer factor analysis, and drug analysis. The review commences by introducing the basic transmission principles, fabrication methods, structural characteristics, detection techniques, and sample pretreatment process of modern lab-on-paper devices. A comprehensive review of the most recent applications of lab-on-paper devices to the diagnosis of common human diseases using blood samples is then presented. The review concludes with a brief summary of the main challenges and opportunities facing the lab-on-paper technology field in the coming years.

Detecting vascular endothelial growth factor C (VEGF-C), a kind of tumor biomarker, is of significant clinical importance in evaluating the prognosis of patients with cancer. However, laboratory analyses are usually not suitable for point-of-care testing because they are expensive and time consuming. In response to these challenges, we fabricated an origami paper-based microfluidic electrochemical device. To improve the specificity of VEGF-C detection, nanocomposites, synthesized by new methylene blue (NMB), amino-functional single-walled carbon nanotubes (NH2-SWCNTs), and gold nanoparticles (AuNPs), were used to modify the surface of working electrodes. Results of electrochemical detection showed that the immunosensor had excellent linearity, ranging from 0.01 to 100 ng mL−1 (R2 = 0.988), and the limit of detection was 10 pg mL−1. To confirm the high specificity of the device under real-world conditions, we evaluated the device using clinical serum samples from our hospital. The results demonstrated that the device had an excellent performance and could provide a platform for real-time detection of cancers., Sun, Wang et al. report an origami paper-based immunosensor for the electrochemical detection of the VEGF-C biomarker in blood serum. The immunosensor is made by modifying the surface of working electrodes with new methylene blue, amino-functional single-walled carbon nanotubes, and gold nanoparticles and demonstrates excellent performance with a limit of detection in the range of picograms per milliliter.

The major obstacle to developing nanozymes which are considered as promising alternatives to natural enzymes is their moderate performance, including poor affinity for substrates, low catalytic activity, and severe pH-dependence. To address these issues, herein, we synthesize ultrathin layered double hydroxide (LDH) nanosheets with a thickness of 1.4 nm and an average lateral size of 23 nm using a fast-precipitation method. Through the rational design of their compositions, it is found that NiMn LDHs exhibit the optimum peroxidase mimicking performance with excellent substrate affinity, high catalytic activity (a limit of detection (LOD) of 0.04 μM H2O2) and robustness in a wide pH range (from 2.6 to 9.0), which is superior to that of natural horseradish peroxidase (HRP). The main active centers are identified as Mn sites because of their strong Lewis acidity and low redox potential. Furthermore, a series of disposable paper bioassays based on NiMn LDH nanozymes are designed and used for the highly sensitive detection of H2O2 and ascorbic acid (AA).

Infectious diseases cause millions of deaths annually in the developing world. Recently, microfluidic paper-based analytical devices (μPADs) have been developed to diagnose such diseases, as these tests are low cost, biocompatible, and simple to fabricate. However, current μPADs are difficult to use in resource-limited areas due to their reliance on external instrumentation to measure and analyze the test results. In this work, we propose an electricity and external instrumentation-free μPAD sensor based on the colorimetric enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infectious disease (3D-tPADs). Designed based on the principle of origami, the proposed μPAD enables the sequential steps of the colorimetric ELISA test to be completed in just ∼10 min. In addition, in order to obtain an accurate ELISA result without using any instrument, we have integrated an electricity-free "timer" within the μPAD that can be controlled by the buffer viscosity and fluid path volume to indicate the appropriate times for washing and color development steps, which can avoid false positive or false negative results caused by an extended or shortened amount of washing and development times. Due to the low background noise and high positive signal intensity of the μPAD, positive and negative detection results can be distinguished by just the naked eye. Furthermore, the ELISA result can be semi-quantified by comparing the results shown on the μPAD with a color chart diagram with a detection limit of HIV type 1(HIV-1) p24 antigen as low as 0.03 ng mL-1. These results demonstrate the proposed sensor can perform infectious disease diagnosis without external instrumentation or electricity, extending the application of the μPAD test for on-site detection and use in resource-limited settings.

The illegal use of β-adrenergic agonists during livestock growth poses a threat to public health; the long-term intake of this medication can cause serious physiological side effects and even death. Therefore, rapid detection methods for β-adrenergic agonist residues on-site are required. Traditional detection methods such as liquid chromatography have limitations in terms of expensive instruments and complex operations. In contrast, paper methods are low cost, ubiquitous, and portable, which has led to them becoming the preferred detection method in recent years. Various paper-based fluidic devices have been developed to detect β-adrenergic agonist residues, including lateral flow immunoassays (LFAs) and microfluidic paper-based analytical devices (μPADs). In this review, the application of LFAs for the detection of β-agonists is summarized comprehensively, focusing on the latest advances in novel labeling and detection strategies. The use of μPADs as an analytical platform has attracted interest over the past decade due to their unique advantages and application for detecting β-adrenergic agonists, which are introduced here. Vertical flow immunoassays are also discussed for their shorter assay time and stronger multiplexing capabilities compared with LFAs. Furthermore, the development direction and prospects for the commercialization of paper-based devices are considered, shedding light on the development of point-of-care testing devices for β-adrenergic agonist residue detection.

In vitro diagnosis (IVD) has become a hot topic in laboratory research and achievement transformation. However, due to the high cost, and time-consuming and complex operation of traditional technologies, some new technologies are being introduced into IVD, to solve the existing problems. As a result, IVD has begun to develop toward point-of-care testing (POCT), a subdivision field of IVD. The pandemic has made governments and health institutions realize the urgency of accelerating the development of POCT. Microfluidic paper-based analytical devices (μPADs), a low-cost, high-efficiency, and easy-to-operate detection platform, have played a significant role in advancing the development of IVD. μPADs are composed of paper as the core material, certain unique substances as reagents for processing the paper, and sensing devices, as auxiliary equipment. The published reviews on the same topic lack a comprehensive and systematic introduction to μPAD classification and research progress in IVD segmentation. In this paper, we first briefly introduce the origin of μPADs and their role in promoting IVD, in the introduction section. Then, processing and detection methods for μPADs are summarized, and the innovative achievements of μPADs in IVD are reviewed. Finally, we discuss and prospect the upgrade and improvement directions of μPADs, in terms of portability, sensitivity, and automation, to help researchers clarify the progress and overcome the difficulties in subsequent μPAD research.

Mineralized polymer scaffolds have proven to be effective biomaterials for inducing osteoinductivity in bone tissue engineering. Sequential mineralization is a promising technique for depositing minerals in three-dimensional (3D) scaffolds. Paper, which is made of cellulose fibers, can be used as a tissue scaffold due to its highly porous structure and flexibility, as well as its excellent ability to wick fluids and support the growth of bone cells. In this study, paper-based, mineralized scaffolds were fabricated using sequential mineralization. We conducted experiments with two groups of scaffolds based on different incubation times in the mineralization solutions (30 min and 24 h). Ten cycles of mineralization were performed for each group. We found that the mineral content increased as the cycle number increased and that the 24-h group scaffolds consistently had more mineralization than did the 30-min group scaffolds when measured at the same cycle number. A quantitative reverse transcription-polymerase chain reaction was performed for two osteogenic differentiation markers of the preosteoblasts that were grown on the mineralized paper scaffolds. The gene expression results for bone-specific markers revealed that the mineralized scaffolds were osteoinductive. Subcutaneous implantation of the scaffolds in rats demonstrated favorable biocompatibility, high vascularization, and non-immunogenicity in vivo. The overall results suggest that the sequentially mineralized paper scaffolds are promising materials for use in bone tissue engineering.

Cotton is cheap, easily available and widely used as surgical material. Therefore, cotton would be good raw material to design a scaffold for tissue engineering applications. In this work, the gelatin-coated microfibrous paper scaffold was fabricated successfully by a papermaking process. Microfibers-based scaffold could overcome the limitations of nanofibrous material-based scaffold for tissue engineering application. The physicochemical and mechanical properties of the scaffolds were characterized. The results revealed that the gelatin contributed to the enhanced cell attachment and proliferation over the scaffolds, whereas cellulose as a strong backbone in the scaffold to support it for keeping its appearance. Their tensile strength and water absorption capacity were improved, but pore size and porosity were decreased after incorporation of gelatin. Hence, results suggested that fabricated scaffolds have huge prospective as a bioactive, well-designed and economical scaffold stand for tissue engineering application.

In the present article we report a new hybrid microfluidic device (hyFlow) comprising a disposable paper electrode and a three-dimensional (3D) printed plastic chip for the electrochemical detection of a magnetic bead-silver nanoparticle (MB-AgNP) bioconjugate. This hybrid device evolved due to the difficulty of incorporating micron-scale MBs into paper-only fluidic devices. Specifically, paper fluidic devices can entrap MB-containing conjugates within their cellulose or nitrocellulose fiber matrix. The hyFlow system was designed to minimize such issues and transport MB conjugates more efficiently to the electrochemical detection zone of the device. The hyFlow system retains the benefit of fluid transport by pressure-driven flow, however, no pump is required for its operation. The hyFlow device is capable of detecting either pre-formed MB-AgNP conjugates or conjugates formed in-situ formation. The detection limit of AgNPs using this device is 12 pM, which represents just 22 AgNPs per MB.

The increasing prevalence of antibiotic-resistant bacteria needs rapid identification and efficient destruction routes. This study proposes testing paper derived from electrospun fibrous mats and aggregation-induced emission (AIE) probes for trace sensing and simultaneous destruction of antibiotic-resistant E. coli. Aptamers are conjugated on fibers for selective capture of E. coli, and the capture capability can be regenerated via rinsing with salt solution. Hydroxyl tetraphenylethene (TPE) is linked with two cephalosporin molecules to construct TPE-Cep probes, and the fluorescence emission is turned on specifically in the presence of β-lactamase, which is a critical marker for screening resistant bacteria. Fibrous mats are lit up only in the presence of antibiotic-resistant bacteria, and the fluorescence intensity changes could be statistically fitted into an equation for quantitative analysis. Fibrous strips display apparent color changes from blue to green for a visual readout of bacterial levels, and the limit of detection (LOD) is much lower than those of previous paper substrates. In addition, the TPE-Cep probes could produce reactive oxygen species (ROS) under room light illumination to kill the captured bacteria. Thus, the integration of aptamer-grafted electrospun fibers and functional AIE probes provides potential for selective capture, trace imaging and photodynamic destruction of antibiotic-resistant bacteria.

Atherosclerosis (AS) is the most common factor causing many cardiovascular and cerebrovascular diseases and has received considerable attention. The occurrence mechanism of AS is uncertain because it is a choronically pathological process that is influenced by multi-aspects, among which cytokines play the key roles in regulating the processes of the immune system. For example, two key cytokines, namely, IL-10 and MCP-1 (chemokine), which are involved in AS progression with varied levels, can be used for AS status monitoring and early diagnosis of AS-associated diseases. Hence, a new paper-based, surface-enhanced Raman spectroscopy (SERS) sensing platform was established for the detection of these two key cytokines. By combining a nanoporous networking membrane as the substrate and SERS nanotags as the probe for signal reading, together with a sandwich design, sensitive and specific identification and quantification of cytokine targets in human serum were achieved with excellent sensing characteristics. The lowest detectable concentration was determined to be 0.1 pg mL-1 for both IL-10 and MCP-1 in human serum. The assay also exhibits high specificity towards target cytokine detection, with low-nonspecific binding and acceptable cross-reactivity in the presence of other structurally similar targets. Finally, the practicability was validated by performing duplex detection in human serum, which further demonstrates the high specificity of the assay for the detection of target cytokines. Taken together, these promising results illustrate that this developed sensing assay is a candidate for clinical multi-target analysis in real environments.

More than 60% newborns experience hyperbilirubinemia and jaundice within the initial week after birth due to the accumulation of total bilirubin in blood. Left untreated high levels of bilirubin may result in brain impairment. Simple, fast, accurate, low-cost and timely point-of-care (POC) analysis of total bilirubin is an unmet need especially in resource-limited areas. This work introduces a novel sensing device, named a "tape-paper sensor", capable of separating plasma from whole blood and measuring total bilirubin by a colorimetric diazotization method. The tape-paper sensing method overcomes non-homogeneous color distribution caused by the "coffee stain" effect, which improves the accuracy of colorimetric evaluation on paper-based analytical devices. The level of hemolysis in the plasma extracted by the device is evaluated, confirming no interference in the detection of total bilirubin. The accuracy of the tape-paper sensing approach for neonatal blood sample measurement is verified by comparison with the hospital pathology laboratory method. The small volume of samples and reagents, minimal equipment (an office scanner), fast detection (

Recently, portable sensing devices with point of care testing (POCT) capability have attracted great attention due to their inherent affordability and accessibility in low resource areas. Paper sensors possess excellent potential as POCT platforms because of low cost, ease of operation, disposability and high-volume manufacturing. Paper sensors that incorporate functional nucleic acids (FNAs) as recognition elements are particularly attractive given that FNAs can be isolated from random-sequence nucleic acid pools to recognize, or respond to, virtually any target of interest. In this review, the advantages of FNAs, particularly DNA aptamers and DNAzymes, as recognition elements for the design of paper sensors are first discussed. This is followed by reviewing three specific types of FNA based paper sensors: dot blots, lateral flow assays, and microfluidic paper-based analytical devices. Furthermore, advances in the signal reporting methods used by FNA based paper sensors are summarized. Finally, limitations of current FNA based paper sensors are discussed along with considerations of future research directions.

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0-5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Objective. Neuroadaptive paradigms that systematically assess event-related potential (ERP) features across many different experimental parameters have the potential to improve the generalizability of ERP findings and may help to accelerate ERP-based biomarker discovery by identifying the exact experimental conditions for which ERPs differ most for a certain clinical population. Obtaining robust and reliable ERPs online is a prerequisite for ERP-based neuroadaptive research. One of the key steps involved is to correctly isolate electroencephalography artifacts in real time because they contribute a large amount of variance that, if not removed, will greatly distort the ERP obtained. Another key factor of concern is the computational cost of the online artifact handling method. This work aims to develop and validate a cost-efficient system to support ERP-based neuroadaptive research. Approach. We developed a simple online artifact handling method, single trial PCA-based artifact removal (SPA), based on variance distribution dichotomies to distinguish between artifacts and neural activity. We then applied this method in an ERP-based neuroadaptive paradigm in which Bayesian optimization was used to search individually optimal inter-stimulus-interval (ISI) that generates ERP with the highest signal-to-noise ratio. Main results. SPA was compared to other offline and online algorithms. The results showed that SPA exhibited good performance in both computational efficiency and preservation of ERP pattern. Based on SPA, the Bayesian optimization procedure was able to quickly find individually optimal ISI. Significance. The current work presents a simple yet highly cost-efficient method that has been validated in its ability to extract ERP, preserve ERP effects, and better support ERP-based neuroadaptive paradigm.

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

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

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

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

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

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

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

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

As enzymes in the outer membrane of the mitochondrion, monoamine oxidases (MAOs) can catalyze the oxidative deamination of monoamines in the human body. According to different substrates, MAOs can be divided into MAO-A and MAO-B. The imbalance of the MAO-A is associated with neurological degeneration, while excess MAO-B activity is closely connected with Parkinson's disease (PD) and Alzheimer's disease (AD); therefore, detection of MAOs is of great significance for the diagnosis and treatment of these diseases. This work reports the multiplexed detection of MAO-A and MAO-B using paper-based devices based on chemiluminescence (CL). The detection limits were 5.01 pg/mL for MAO-A and 8.50 pg/mL for MAO-B in human serum. In addition, we used paper-based devices to detect MAOs in human cells and tissue samples and found that the results of paper-based detection and Western blotting (WB) showed the same trend. While only one antibody can be incubated on the same membrane by WB, multiple antibodies incubated on the same paper enabled simultaneous detection of MAO-A and MAO-B by paper-based devices. The paper-based assay could be used for preliminary early screening of clinical samples for MAOs and can be extended as an alternative to WB for multiplexed detection of various proteins in disease cell or tissue samples.

Significance: Diffuse reflectance spectroscopy (DRS) allows discrimination of tissue type. Its application is limited by the inability to mark the scanned tissue and the lack of real-time measurements. Aim: This study aimed to develop a real-time tracking system to enable localization of a DRS probe to aid the classification of tumor and non-tumor tissue. Approach: A green-colored marker attached to the DRS probe was detected using hue-saturation-value (HSV) segmentation. A live, augmented view of tracked optical biopsy sites was recorded in real time. Supervised classifiers were evaluated in terms of sensitivity, specificity, and overall accuracy. A developed software was used for data collection, processing, and statistical analysis. Results: The measured root mean square error (RMSE) of DRS probe tip tracking was 1.18±0.58 mm and 1.05±0.28 mm for the x and y dimensions, respectively. The diagnostic accuracy of the system to classify tumor and non-tumor tissue in real time was 94% for stomach and 96% for the esophagus. Conclusions: We have successfully developed a real-time tracking and classification system for a DRS probe. When used on stomach and esophageal tissue for tumor detection, the accuracy derived demonstrates the strength and clinical value of the technique to aid margin assessment in cancer resection surgery.

We report a colorimetric paper-based microfluidic device based on an enzyme inhibition assay that allows the on-site detection of nerve agents by sampling and wicking. The sample and reagents are automatically transported through the channel where an enzyme inhibition reaction is conducted, followed by an enzyme-substrate reaction and a color reaction. This device can detect 0.1 μg/mL of the nerve agent VX in a 2.5 μL drop and is nerve agent selective and robust against temperature, pH, and several liquids. We confirmed that sampling procedures (dilution and wiping) are applicable to this device. Furthermore, the fabrication procedure is easy, and the cost is at most a few tens of cents. Thus, the present device provides a practical method for the urgent detection of nerve agents in suspected chemical terrorism incidents.

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

Significance: Tissue-like solid phantoms with identical optical properties, known within tolerant uncertainty, are of crucial importance in diffuse optics for instrumentation assessment, interlaboratory comparison studies, industrial standards, and multicentric clinical trials. Aim: The reproducibility in fabrication of homogeneous solid phantoms is focused based on spectra measurements by instrument comparisons grounded on the time-resolved diffuse optics. Approach: Epoxy-resin and silicone phantoms are considered as matrices and both employ three different instruments for time-resolved diffuse spectroscopy within the spectral range of 540 to 1100 nm. In particular, we fabricated two batches of five phantoms each in epoxy resin and silicone. Then, we evaluated the intra- and interbatch variability with respect to the instrument precision, by considering the coefficient of variation (CV) of absorption and reduced scattering coefficients. Results: We observed a similar precision for the three instruments, within 2% for repeated measurements on the same phantom. For epoxy-resin phantoms, the intra- and the interbatch variability reached the instrument precision limit, demonstrating a very good phantom reproducibility. For the silicone phantoms, we observed larger values for intra- and interbatch variability. In particular, at worst, for reduced scattering coefficient interbatch CV was about 5%. Conclusions: Results suggest that the fabrication of solid phantoms, especially considering epoxy-resin matrix, is highly reproducible, even if they come from different batch fabrications and are measured using different instruments.

Journal of neural engineering 19(3), 036045 (2022). doi:10.1088/1741-2552/ac77c0, Published by Institute of Physics Publishing, Bristol

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.

This research has developed a method for rapid detection of SARS-CoV-2 N protein on a paper-based microfluidic chip. The chitosan-glutaraldehyde cross-linking method is used to fix the coated antibody, and the sandwich enzyme-linked immunosorbent method is used to achieve the specific detection of the target antigen. The system studied the influence of coating antibody concentration and enzyme-labeled antibody concentration on target antigen detection. According to the average gray value measured under different N protein concentrations, the standard curve of the method was established and the sensitivity was tested, and its linear regression was obtained. The equation is y = 9.8286x+137.6, R2 = 0.9772 > 0.90, which shows a high degree of fit. When the concentration of coating antibody and enzyme-labeled antibody were 1 μg/mL and 2 μg/mL, P > 0.05, the difference was not statistically significant, so the lower concentration of 1 μg/mL was chosen as the coating antibody concentration. The results show that the minimum concentration of N protein that can be detected by this method is 8 μg/mL, and the minimum concentration of coating antibody and enzyme-labeled antibody is 1 μg/mL, which has the characteristics of high sensitivity and good repeatability.

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

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

Significance: During the development and early testing phases of new photoacoustic (PA) breast imaging systems, several choices need to be made in aspects of system design and measurement sequences. Decision-making can be complex for state-of-the-art systems such as 3D hybrid photoacoustic-ultrasound (PA-US) breast imagers intended for multispectral quantitative imaging. These systems have a large set of design choices and system settings that affect imaging performance in different ways and often require trade-offs. Decisions have to be made carefully as they can strongly influence the imaging performance. Aim: A systematic approach to assess the influence of various choices on the imaging performance in carefully controlled laboratory situations is crucial before starting with human studies. Test objects and phantoms are used for first imaging studies, but most reported structures have a 2D geometry and are not suitable to assess all the image quality characteristics (IQCs) of 3D hybrid PA-US systems. Approach: Our work introduces a suite of five test objects designed for hybrid PA-US systems with a 3D detection aperture. We present the test object designs and production protocols and explain how they can be used to study various performance measures. To demonstrate the utility of the developed objects, measurements are made with an existing tomographic PA system. Results: Two test objects were developed for measurements of the US detectors’ impulse responses and light distribution on the breast surface. Three others were developed to assess image quality and quantitative accuracy of the PA and US modes. Three of the five objects were imaged to demonstrate their use. Conclusions: The developed test objects allow one to study influences of various choices in design and system settings. With this, IQCs can be assessed as a function of measurement sequence settings for the PA and US modes in a controlled way. Systematic studies and measurements using these objects will help to optimize various system settings and measurement protocols in laboratory situations before embarking on human studies.

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