Assessing cell viability with dynamic optical coherence microscopy.

Assessing cell viability is important in many fields of research. Current optical methods to assess cell viability typically involve fluorescent dyes, which are often less reliable and have poor permeability in primary tissues. Dynamic optical coherence microscopy (dOCM) is an emerging tool that provides label-free contrast reflecting changes in cellular metabolism. In this work, we compare the live contrast obtained from dOCM to viability dyes, and for the first time to our knowledge, demonstrate that dOCM can distinguish live cells from dead cells in murine syngeneic tumors. We further demonstrate a strong correlation between dOCM live contrast and optical redox ratio by metabolic imaging in primary mouse liver tissue. The dOCM technique opens a new avenue to apply label-free imaging to assess the effects of immuno-oncology agents, targeted therapies, chemotherapy, and cell therapies using live tumor tissues.

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array.

Significance: Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. Aim: We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. Approach: A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. Results: An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30× to 6× acquisition speed advantage for the light sheet compared to the laser scanning geometry. Conclusions: FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array.

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

Multiscale Label-Free Imaging of Fibrillar Collagen in the Tumor Microenvironment.

With recent advances in cancer therapeutics, there is a great need for improved imaging methods for characterizing cancer onset and progression in a quantitative and actionable way. Collagen, the most abundant extracellular matrix protein in the tumor microenvironment (and the body in general), plays a multifaceted role, both hindering and promoting cancer invasion and progression. Collagen deposition can defend the tumor with immunosuppressive effects, while aligned collagen fiber structures can enable tumor cell migration, aiding invasion and metastasis. Given the complex role of collagen fiber organization and topology, imaging has been a tool of choice to characterize these changes on multiple spatial scales, from the organ and tumor scale to cellular and subcellular level. Macroscale density already aids in the detection and diagnosis of solid cancers, but progress is being made to integrate finer microscale features into the process. Here we review imaging modalities ranging from optical methods of second harmonic generation (SHG), polarized light microscopy (PLM), and optical coherence tomography (OCT) to the medical imaging approaches of ultrasound and magnetic resonance imaging (MRI). These methods have enabled scientists and clinicians to better understand the impact collagen structure has on the tumor environment, at both the bulk scale (density) and microscale (fibrillar structure) levels. We focus on imaging methods with the potential to both examine the collagen structure in as natural a state as possible and still be clinically amenable, with an emphasis on label-free strategies, exploiting intrinsic optical properties of collagen fibers.

Measuring the spatial distribution of multiply scattered light using a de-scanned image sensor for examining retinal structure contrast.

An optical platform is presented for examining intrinsic contrast detection strategies when imaging retinal structure using ex vivo tissue. A custom microscope was developed that scans intact tissue and collects scattered light distribution at every image pixel, allowing digital masks to be applied after image collection. With this novel approach at measuring the spatial distribution of multiply scattered light, known and novel methods of detecting intrinsic cellular contrast can be explored, compared, and optimized for retinal structures of interest.

Calibration of liquid crystal variable retarders using a common-path interferometer and fit of a closed-form expression for the retardance curve.

A liquid crystal variable retarder (LCVR) enables fast, automated control of retardance that can be used as a variable waveplate in polarimetric instruments. However, precise control of the polarization state requires calibration of the LCVR. A manufacturer calibration curve is typically supplied for a single specific wavelength and temperature, but for applications under different conditions, additional calibration is needed. Calibration is typically performed with crossed polarizers to generate an intensity curve that is converted to retardance, but this method is prone to noise when retardance is close to zero. Here, we demonstrate a simple common-path Sagnac interferometer to measure retardance and provide open source software for automated generation of calibration curves for retardance as a function of wavelength and voltage. We also provide a curve fitting method and closed-form functional representation that outputs the voltage needed to achieve a desired retardance given a specified wavelength.

Platform for quantitative multiscale imaging of tissue composition.

Changes in the multi-level physical structure of biological features going from cellular to tissue level composition is a key factor in many major diseases. However, we are only beginning to understand the role of these structural changes because there are few dedicated multiscale imaging platforms with sensitivity at both the cellular and macrostructural spatial scale. A single platform reduces bias and complications from multiple sample preparation methods and can ease image registration. In order to address these needs, we have developed a multiscale imaging system using a range of imaging modalities sensitive to tissue composition: Ultrasound, Second Harmonic Generation Microscopy, Multiphoton Microscopy, Optical Coherence Tomography, and Enhanced Backscattering. This paper details the system design, the calibration for each modality, and a demonstration experiment imaging a rabbit eye.

Noise reduction in supercontinuum sources for OCT by single-pulse spectral normalization.

Supercontinuum (SC) sources offer high illumination power from a single mode fiber with large spectral bandwidth including the visible spectrum, a growing application area for Optical Coherence Tomography (OCT). However, SC spectra suffer from pulse-to-pulse variations, increasing noise in the resulting images. By simultaneously collecting a normalization spectrum, OCT image noise can be reduced by more than half (7 dB) for single pulses without any pulse averaging using only simple optical components.

The advance care planning nurse facilitator: describing the role and identifying factors associated with successful implementation.

Advance care planning (ACP) has been shown to improve end-of-life care, yet uptake remains limited. Interventions aimed at increasing ACP uptake have often used a 'specialist ACP facilitator' model. The present qualitative study appraised the components of an ACP facilitator intervention comprising nurse-led patient screening and ACP discussions, as well as factors associated with the successful implementation of this model in primary care and acute hospital settings across rural and metropolitan Western Australia. Semistructured interviews were undertaken with 17 health professionals who were directly or indirectly involved in the facilitator ACP intervention among patients with severe respiratory disease. Additional process data (nurse facilitator role description, agreements with participating sites) were used to describe the nurse facilitator role. The interview data identified factors associated with successful implementation, including patient factors, health professional factors, ACP facilitator characteristics and the optimal settings for the intervention. The primary care setting was seen as most appropriate, and time limitations were a key consideration. Factors associated with successful implementation included trusting relationships between the nurse facilitator and referring doctor, as well as opportunities for meaningful encounters with patients. This study suggests a model of ACP nurse facilitation based in primary care may be an acceptable and effective method of increasing ACP uptake.

Single-particle photothermal imaging via inverted excitation through high-Q all-glass toroidal microresonators.

Whispering-gallery mode (WGM) microresonators have recently been employed as platforms for label-free single-molecule and single-particle detection, imaging, and spectroscopy. However, innovations in device geometry and integration are needed to make WGM microresonators more versatile for biological and chemical applications. Particularly, thick device substrates, originating from wafer-scale fabrication processing, prevent convenient optical interrogation. In this work, we fabricate all-glass toroidal microresonators on a coverslip thickness (~170 µm) substrate, enabling excitation delivery through the sample, simplifying optical integration. Further, we demonstrate the application of this new geometry for single-particle photothermal imaging. Finally, we discover and develop simulations to explain a non-trivial astigmatism in the point spread function (PSF) arising from the curvature of the resonator.

Quantifying optical properties with visible and near-infrared optical coherence tomography to visualize esophageal microwave ablation zones.

Microwave ablation is a minimally invasive image guided thermal therapy for cancer that can be adapted to endoscope use in the gastrointestinal (GI) tract. Microwave ablation in the GI tract requires precise control over the ablation zone that could be guided by high resolution imaging with quantitative contrast. Optical coherence tomography (OCT) provides ideal imaging resolution and allows for the quantification of tissue scattering properties to characterize ablated tissue. Visible and near-infrared OCT image analysis demonstrated increased scattering coefficients (µs ) in ablated versus normal tissues (Vis: 347.8%, NIR: 415.0%) and shows the potential for both wavelength ranges to provide quantitative contrast. These data suggest OCT could provide quantitative image guidance and valuable information about antenna performance in vivo.

Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration.

PURPOSE: We investigated the autofluorescence (AF) signature of the microscopic features of retina with age-related macular degeneration (AMD) using 488 nm excitation. METHODS: The globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496-610 nm, 6 nm resolution). The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch's Membrane, as well macroscopic features, were considered. RESULTS: Overall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch's Membrane across all the tissue cross-sections (p = 0.02). CONCLUSIONS: The AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation) compared with the control tissue. The magnitude of these differences is small (~4 nm) and highlights the potential challenges of detecting these subtle spectral differences in vivo.

Pancreatic ß-Cells From Mice Offset Age-Associated Mitochondrial Deficiency With Reduced KATP Channel Activity.

Aging is accompanied by impaired glucose homeostasis and an increased risk of type 2 diabetes, culminating in the failure of insulin secretion from pancreatic ß-cells. To investigate the effects of age on ß-cell metabolism, we established a novel assay to directly image islet metabolism with NAD(P)H fluorescence lifetime imaging (FLIM). We determined that impaired mitochondrial activity underlies an age-dependent loss of insulin secretion in human islets. NAD(P)H FLIM revealed a comparable decline in mitochondrial function in the pancreatic islets of aged mice (≥24 months), the result of 52% and 57% defects in flux through complex I and II, respectively, of the electron transport chain. However, insulin secretion and glucose tolerance are preserved in aged mouse islets by the heightened metabolic sensitivity of the ß-cell triggering pathway, an adaptation clearly encoded in the metabolic and Ca(2+) oscillations that trigger insulin release (Ca(2+) plateau fraction: young 0.211 ± 0.006, aged 0.380 ± 0.007, P < 0.0001). This enhanced sensitivity is driven by a reduction in KATP channel conductance (diazoxide: young 5.1 ± 0.2 nS; aged 3.5 ± 0.5 nS, P < 0.01), resulting in an ∼2.8 mmol/L left shift in the ß-cell glucose threshold. The results demonstrate how mice but not humans are able to successfully compensate for age-associated metabolic dysfunction by adjusting ß-cell glucose sensitivity and highlight an essential mechanism for ensuring the maintenance of insulin secretion.

How Well Do We Understand Health Care Professionals' Perceptions and Needs in the Provision of Palliative Care? A Mixed Methods Study.

OBJECTIVES: Despite palliative care being standard for patients with chronic and/or life-limiting conditions, a perceived lack of clarity regarding the definition and scope of palliative care persists. We aimed to identify health care professionals' (HCPs) perspectives, education, and support needs related to palliative care provision in a large private Australian tertiary hospital. METHODS: A validated survey was administered and four focus groups were conducted with multidisciplinary HCPs. RESULTS: The survey response rate was 50% (n = 302). Although critical care HCPs scored symptom management and patient/family interaction items more highly compared with other HCPs, mean scores (<4.0) for both groups indicated participants lacked confidence to perform this aspect of care independently. Critical care HCPs were more comfortable caring for dying patients (p < 0.001) and talking to families about death (p < 0.001). Ward HCPs were more supportive of early referral to palliative care (p < 0.001). Cancer diagnoses were overestimated as common causes of death. Education needs focused on ethical issues, end-of-life communication skills, dealing with delirium, and use of the Liverpool Care Pathway. Key themes identified from the four focus groups were (1) delays or nonreferral to palliative care created considerable stress and feelings of inadequacy despite a perceived understanding of the broader definition of palliative care and (2) HCPs commonly focused on end-of-life care. CONCLUSION: Ambiguity regarding the meaning and delivery of palliative care persists in the acute care setting across disciplines. Results confirmed that innovative approaches to education and upskilling HCPs in palliative care and referral pathways is warranted.

REACTED - Reducing Acute Chest pain Time in the ED: A prospective pre-/post-interventional cohort study, stratifying risk using early cardiac multi-markers, probably increases discharges safely.

OBJECTIVE: ED chest pain assessments can be challenging, lengthy and contribute to overcrowding. Rapid accurate risk stratification strategies should improve ED length of stay (EDLOS). Emergency, Biochemistry and Cardiology implemented new guidelines using paired (<3 h) multiple cardiac markers to stratify patients. The intervention would reduce chest pain EDLOS. We observed for safety and disposition effects. METHODS: This is a single-site, prospective observational, before and after intervention study. In December 2009, paired multiple cardiac markers, the second at least 4 h from pain, replaced late troponins. The 4 h rule (ED flow improvement) started in April 2009 (unplanned confounder). Demographics, clinical features, risk assessment and disposition; preferably prospective. Administrative datasets provided disposition outcomes, 4 months pre-/post-intervention. Follow up with partially blinded adjudications assessed for 45 day major adverse cardiac events (MACE). Before intervention, consecutive patients were enrolled with mixed prospective/retrospective data. After, sampling occurred whenever prospective data were collected. RESULTS: Adjudicated patients were n = 1029 (14.2% MI, 14.9% MACE), 426 before, 603 after. EDLOS reduced 87 min (416-329; P < 0.001), similar to triage 2 patients without chest pain. Possibly, avoidable MACE occurred in five of 598 discharges (0.8%, CI 0.3-1.8%) with non-significant decreases (0.5%, CI 0.1-1.8%) post-intervention. Disposition changes included greater observation ward use (3.8-12.3%; P < 0.001), more discharges (47.4-52.9%, P = 0.002), less transfers (9.3-6.9%, P = 0.014) and less late inpatient discharge decisions (15.2-8.7%, P = 0.001). CONCLUSION: Paired cardiac markers performed adequately for avoidable MACE, and disposition improved significantly. A confounding system change meant the reduced EDLOS (primary outcome) was unable to be associated with the intervention.

Modeling Study of a Proposed Field Calibration Source Using K-40 and High-Z Targets for Sodium Iodide Detectors.

Calibration sources based on the primordial isotope potassium-40 (K) have reduced controls on the source's activity due to its terrestrial ubiquity and very low specific activity. Potassium-40's beta emissions and 1,460.8 keV gamma ray can be used to induce K-shell fluorescence x rays in high-Z metals between 60 and 80 keV. A gamma ray calibration source that uses potassium chloride salt and a high-Z metal to create a two-point calibration for a sodium iodide field gamma spectroscopy instrument is thus proposed. The calibration source was designed in collaboration with the Sandia National Laboratory using the Monte Carlo N-Particle eXtended (MCNPX) transport code. Two methods of x-ray production were explored. First, a thin high-Z layer (HZL) was interposed between the detector and the potassium chloride-urethane source matrix. Second, bismuth metal powder was homogeneously mixed with a urethane binding agent to form a potassium chloride-bismuth matrix (KBM). The bismuth-based source was selected as the development model because it is inexpensive, nontoxic, and outperforms the high-Z layer method in simulation. Based on the MCNPX studies, sealing a mixture of bismuth powder and potassium chloride into a thin plastic case could provide a light, inexpensive field calibration source.

Skeletal light-scattering accelerates bleaching response in reef-building corals.

BACKGROUND: At the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 µm layer of corals skeletons (measured as 'microscopic' reduced-scattering coefficient, µ'(S,m)), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species. RESULTS: Here we experimentally demonstrate (in ten coral species) that, under thermal stress alone or combined thermal and light stress, low-µ'(S,m) corals bleach at higher rate and severity than high-µ'(S,m) corals and the Symbiodinium associated with low-µ'(S,m) corals experience twice the decrease in photochemical efficiency. We further modelled the light absorbed by Symbiodinium due to skeletal-scattering and show that the estimated skeleton-dependent light absorbed by Symbiodinium (per unit of photosynthetic pigment) and the temporal rate of increase in absorbed light during bleaching are several fold higher in low-µ'(S,m) corals. CONCLUSIONS: While symbionts associated with low-[Formula: see text] corals receive less total light from the skeleton, they experience a higher rate of light increase once bleaching is initiated and absorbing bodies are lost; further precipitating the bleaching response. Because microscopic skeletal light-scattering is a robust predictor of light-dependent bleaching among the corals assessed here, this work establishes µ'(S,m) as one of the key determinants of differential bleaching response.

Emergency department 'undercrowding' is associated with decreased waiting times.

OBJECTIVE: To evaluate the effect of a sudden and sustained decrease in patient presentations on waiting times and other measures of workload and flow following the opening of a large, greenfields ED adjacent to our own. METHOD: A descriptive study involving all patients presenting to a private urban district hospital ED for two 60 day periods, immediately before and after the opening of the tertiary hospital ED. Changes in median waiting time, case-mix distribution, method of arrival, total admissions and total waiting time were compared pre-opening and post-opening. Non-normally distributed variables were analysed using Mann-Whitney U-tests. Categorical variables were compared using χ(2) analyses. RESULTS: Patient presentations decreased by 28% with a parallel decline in median waiting time of 15 min (from 26 to 11 min) (P < 0.001). Total waiting time was approximately 29 h less per day in the post-opening period. Patient urgency by triage category did not change significantly (P = 0.316), whereas the proportion of presentations by ambulance decreased 15.9% (P = 0.048) and admission rate increased from 29.1% to 32.6% (P = 0.002). CONCLUSIONS: Patient presentation numbers are strongly associated with and likely impact on median waiting time. Understanding that controlling demand can lead to significant benefits in patient processing, flow and overall patient perceived level of care and satisfaction is relevant to any discussion on ED overcrowding and the deleterious effects of access block.

Acetylfentanyl: An Emerging Drug of Abuse.

BACKGROUND: Opioid analgesics are widely used in health care, yet have significant potential for abuse. High doses are associated with potentially fatal respiratory depression, which caused 21,314 deaths in the United States in 2011. Acetylfentanyl, a synthetic opioid agonist closely related to fentanyl, recently emerged as a drug of abuse linked to numerous deaths in North America. CASE REPORT: A 36-year-old male developed the habit of using a propylene glycol electronic cigarette filled with acetylfentanyl to aid relaxation. He purchased the drug online in a manner that appeared legal to him, which compromised his insight about the danger of the substance. He had been using the e-cigarette with increasing frequency while on medical leave, and his wife reported finding him weakly responsive on more than one occasion. At approximately 3 am, the family activated 911 for altered mental status. His presentation included respiratory depression, pinpoint pupils, hypoxemia, and a Glasgow Coma Scale score of 6. He responded to serial doses of intravenous naloxone with improvement in his mental status and respiratory condition. Due to the need for repeated dosing, he was placed on a naloxone infusion and recovered uneventfully in intensive care. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Complications from emerging drugs of abuse, like acetylfentanyl, frequently present first to emergency departments. Prompt recognition and treatment can help avoid morbidity and mortality. Acetylfentanyl can be managed effectively with naloxone, although higher than conventional dosing may be required to achieve therapeutic effect.