High resolution TCSPC imaging of diffuse light with a one-dimensional SPAD array scanning system.

We report a time-correlated single-photon counting (TCSPC) imaging system based on a line-scanning architecture. The system benefits from the high fill-factor, active area, and large dimension of an advanced CMOS single-photon avalanche diode (SPAD) array line-sensor. A two-dimensional image is constructed using a moving mirror to scan the line-sensor field-of-view (FOV) across the target, to enable the efficient acquisition of a two-dimensional 0.26 Mpixel TCSPC image. We demonstrate the capabilities of the system for TCSPC imaging and locating objects obscured in scattering media - specifically to locate a series of discrete point sources of light along an optical fibre submerged in a highly scattering solution. We demonstrate that by selectively imaging using early arriving photons which have undergone less scattering than later arriving photons, our TCSPC imaging system is able to locate the position of discrete point sources of light than a non-time-resolved imaging system.

Time-resolved spectroscopy at 19,000 lines per second using a CMOS SPAD line array enables advanced biophotonics applications.

A SPAD-based line sensor fabricated in 130 nm CMOS technology capable of acquiring time-resolved fluorescence spectra (TRFS) in 8.3 milliseconds is presented. To the best of our knowledge, this is the fastest time correlated single photon counting (TCSPC) TRFS acquisition reported to date. The line sensor is an upgrade to our prior work and incorporates: i) parallelized interface from sensor to surrounding circuitry enabling high line rate to the PC (19,000 lines/s) and ii) novel time-gating architecture where detected photons in the OFF region are rejected digitally after the output stage of the SPAD. The time-gating architecture was chosen to avoid electrical transients on the SPAD high voltage supplies when gating is achieved by excess bias modulation. The time-gate has an adjustable location and time window width allowing the user to focus on time-events of interest. On-chip integrated center-of-mass (CMM) calculations provide efficient acquisition of photon arrivals and direct lifetime estimation of fluorescence decays. Furthermore, any of the SPC, TCSPC and on-chip CMM modes can be used in conjunction with the time-gating. The higher readout rate and versatile architecture greatly empower the user and will allow widespread applications across many techniques and disciplines. Here we focused on 3 examples of TRFS and time-gated Raman spectroscopy: i) kinetics of chlorophyll A fluorescence from an intact leaf; ii) kinetics of a thrombin biosensor FRET probe from quenched to fluorescence states; iii) ex vivo mouse lung tissue autofluorescence TRFS; iv) time-gated Raman spectroscopy of toluene at 3056 cm-1 peak. To the best of our knowledge, we detect spectrally for the first time the fast rise in fluorescence lifetime of chlorophyll A in a measurement over single fluorescent transient.

pH sensing through a single optical fibre using SERS and CMOS SPAD line arrays.

Full exploitation of fibre Raman probes has been limited by the obstruction of weak Raman signals by background fluorescence of the sample and the intrinsic Raman signal of the delivery fibre. Here we utilised functionalised gold nanoshells (NS) to take advantage of the surface-enhanced Raman spectroscopy (SERS) effect to enhance the pH responsive spectrum of 4-mercaptobenzoic acid (MBA). However, the fibre background is still dominant. Using the photon arrival time-resolving capability of a CMOS single-photon avalanche diode (SPAD) based line sensor, we recover the SERS spectrum without a fibre background in a 10 s measurement. In this manner, pH sensing through a multimode fibre at a low excitation power that is safe for future in vivo applications, with short acquisition times (10 or 60 s), is demonstrated. A measurement precision of ± 0.07 pH units is thus achieved.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus.

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17ß (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

Combined dysfunctions of immune cells predict nosocomial infection in critically ill patients.

BACKGROUND: Nosocomial infection occurs commonly in intensive care units (ICUs). Although critical illness is associated with immune activation, the prevalence of nosocomial infections suggests concomitant immune suppression. This study examined the temporal occurrence of immune dysfunction across three immune cell types, and their relationship with the development of nosocomial infection. METHODS: A prospective observational cohort study was undertaken in a teaching hospital general ICU. Critically ill patients were recruited and underwent serial examination of immune status, namely percentage regulatory T-cells (Tregs), monocyte deactivation (by expression) and neutrophil dysfunction (by CD88 expression). The occurrence of nosocomial infection was determined using pre-defined, objective criteria. RESULTS: Ninety-six patients were recruited, of whom 95 had data available for analysis. Relative to healthy controls, percentage Tregs were elevated 6-10 days after admission, while monocyte HLA-DR and neutrophil CD88 showed broader depression across time points measured. Thirty-three patients (35%) developed nosocomial infection, and patients developing nosocomial infection showed significantly greater immune dysfunction by the measures used. Tregs and neutrophil dysfunction remained significantly predictive of infection in a Cox hazards model correcting for time effects and clinical confounders {hazard ratio (HR) 2.4 [95% confidence interval (CI) 1.1-5.4] and 6.9 (95% CI 1.6-30), respectively, P=0.001}. Cumulative immune dysfunction resulted in a progressive risk of infection, rising from no cases in patients with no dysfunction to 75% of patients with dysfunction of all three cell types (P=0.0004). CONCLUSIONS: Dysfunctions of T-cells, monocytes, and neutrophils predict acquisition of nosocomial infection, and combine additively to stratify risk of nosocomial infection in the critically ill.

Pulmonary and systemic effects of mononuclear leukapheresis.

BACKGROUND AND OBJECTIVES: There is increasing evidence that monocytes play a key role in the pathogenesis of acute lung inflammation. Mononuclear cell (MNC) leukapheresis can be used to remove large numbers of monocytes from circulating blood; however, the detailed characteristics of monocyte subpopulations removed by MNC leukapheresis, and the biological effects on the lung, remain incompletely defined. MATERIAL AND METHODS: Six healthy male volunteers underwent MNC leukapheresis of four total blood volumes. Blood was collected at 0, 2, 4, 6, 8 and 24 h; bronchoscopy with bronchoalveolar lavage (BAL) was performed at 8-9 h. Multiparameter flow cytometry was used to identify subpopulations of monocytes in blood and monocyte-like cells in BAL fluid. RESULTS: A median of 5·57×10(9) monocytes were retrieved. Blood monocyte counts indicated that the circulating blood monocyte pool was actively replenished during leukapheresis and subsequently contained a greater proportion of classical (CD14(++) CD16(-)) monocytes. A particular subpopulation of monocyte-like cells, reminiscent of classical monocytes, was also prominent in BAL fluid after leukapheresis. CONCLUSION: Mononuclear cell leukapheresis was safe. The greater proportion of classical monocytes present in blood after MNC leukapheresis may be clinically significant. MNC leukapheresis also appears to affect the proportion of monocyte-like cells in the lung; however, we found no evidence that leukapheresis has a clinically important pro-inflammatory effect in the human lung.

Diagnosing ventilator-associated pneumonia (VAP) in UK NHS ICUs: the perceived value and role of a novel optical technology.

BACKGROUND: Diagnosing ventilator-associated pneumonia (VAP) in an intensive care unit (ICU) is a complex process. Our aim was to collect, evaluate and represent the information relating to current clinical practice for the diagnosis of VAP in UK NHS ICUs, and to explore the potential value and role of a novel diagnostic for VAP, which uses optical molecular alveoscopy to visualise the alveolar space. METHODS: Qualitative study performing semi-structured interviews with clinical experts. Interviews were recorded, transcribed, and thematically analysed. A flow diagram of the VAP patient pathway was elicited and validated with the expert interviewees. Fourteen clinicians were interviewed from a range of UK NHS hospitals: 12 ICU consultants, 1 professor of respiratory medicine and 1 professor of critical care. RESULTS: Five themes were identified, relating to [1] current practice for the diagnosis of VAP, [2] current clinical need in VAP diagnostics, [3] the potential value and role of the technology, [4] the barriers to adoption and [5] the evidence requirements for the technology, to help facilitate a successful adoption. These themes indicated that diagnosis of VAP is extremely difficult, as is the decision to stop antibiotic treatment. The analysis revealed that there is a clinical need for a diagnostic that provides an accurate and timely diagnosis of the causative pathogen, without the long delays associated with return of culture results, and which is not dangerous to the patient. It was determined that the technology would satisfy important aspects of this clinical need for diagnosing VAP (and pneumonia, more generally), but would require further evidence on safety and efficacy in the patient population to facilitate adoption. CONCLUSIONS: Care pathway analysis performed in this study was deemed accurate and representative of current practice for diagnosing VAP in a UK ICU as determined by relevant clinical experts, and explored the value and role of a novel diagnostic, which uses optical technology, and could streamline the diagnostic pathway for VAP and other pneumonias.

Evaluation of intranasal nafamostat or camostat for SARS-CoV-2 chemoprophylaxis in Syrian golden hamsters.

Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.

Surgical margins for borderline and malignant phyllodes tumours.

BACKGROUND: Phyllodes tumours represent less than 1% of all UK breast neoplasms. Histological features allow classification into benign, borderline or malignant, which has a significant impact on prognosis and recurrence. Currently, there is no consensus for the optimal surgical excision margin. This systematic review aims to provide a comparative summary of outcomes (local recurrence, metastasis and survival) for borderline and malignant phyllodes tumours resected with either ≥1cm or <1cm margins. METHODS: MEDLINE and Embase were systematically searched (1990 to July 2019), in line with PRISMA guidelines. Study quality was assessed using the Newcastle-Ottawa scale. RESULTS: Ten retrospective studies were included (Newcastle-Ottawa scale mean score: 5.6, range: 8-4). Nine reported local recurrence rates, four reported distant metastasis and four reported survival. Meta-analysis pooling demonstrated no statistically significant difference between <1cm and ≥1cm margins in terms of local recurrence rates (relative risk [RR] 1.43, 95% confidence interval [95% CI] 0.70 - 2.93; p=0.33, n=456), distant metastasis (RR 1.93, 95% CI 0.35 - 10.63; p=0.45, n=72) or mortality (RR 1.93, 95% CI 0.42 - 8.77; p=0.40, n=58) for borderline and malignant tumours. Additionally, two studies demonstrated no significant difference in local recurrence for borderline tumours excised with <0.1cm margins compared to ≥1cm. CONCLUSION: Current evidence suggests that margins <1cm may provide adequate tumour excision. This could enable breast conservation in patients with smaller breast-to-tumour volume ratios, with improved cosmetic outcomes and patient satisfaction. A prospective, multi-institutional trial would be appropriate to further elucidate the safety of smaller margins.

Ballistic and snake photon imaging for locating optical endomicroscopy fibres.

We demonstrate determination of the location of the distal-end of a fibre-optic device deep in tissue through the imaging of ballistic and snake photons using a time resolved single-photon detector array. The fibre was imaged with centimetre resolution, within clinically relevant settings and models. This technique can overcome the limitations imposed by tissue scattering in optically determining the in vivo location of fibre-optic medical instruments.

Endoscopic sensing of alveolar pH.

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.

Review of the cellular and biological principles of distraction osteogenesis: An in vivo bioreactor tissue engineering model.

Distraction osteogenesis (DO) is a widely used technique in plastic and orthopaedic surgery. During the process, mechanical force is applied to fractured bone to enhance the regenerative processes and induce new bone formation. Although there is an abundance of literature on the clinical process of DO, there is a distinct lack of focus on the underlying biological principles governing this process. DO follows the basic premises of tissue engineering. The mechanical stress stimulates mesenchymal stem cell differentiation down an osteoblastic lineage on a matrix background. The aim of this review is to give an overview of the current knowledge of the molecular mechanism governing this process.

A prospective study of the use of botulinum toxin injections in the treatment of Raynaud's syndrome associated with scleroderma.

Raynaud's syndrome contributes to the pain, paraesthesia, ulceration, and gangrene of scleroderma. Botulinum toxin has been shown to improve digital perfusion in patients with Raynaud's. This is the first study to objectively assess hand function following this treatment in patients with scleroderma. Twenty patients were treated with 100 units of botulinum toxin injected into the hand. An assessment of hand function and symptoms was performed prior to injection and then 8-12 weeks later. The outcomes assessed were change in pain, appearance, cold intolerance, pinch and power grip, ranges of movement, and Disabilities of the Arm, Shoulder and Hand (DASH) score. In total, 80% of patients reported an overall improvement in their symptoms, reduction in pain, and improved DASH score and 65% reported improvement in cold intolerance. Overall, 90% showed an improvement in pinch grip and 65% an improvement in power grip. Objective parameters were statistically significantly improved; however, subjective outcomes only showed a trend. We have found botulinum toxin to be an effective treatment for Raynaud's syndrome secondary to scleroderma.

Imaging inflammation: molecular strategies to visualize key components of the inflammatory cascade, from initiation to resolution.

Dysregulation of inflammation is central to the pathogenesis of innumerable human diseases. Understanding and tracking the critical events in inflammation are crucial for disease monitoring and pharmacological drug discovery and development. Recent progress in molecular imaging has provided novel insights into spatial associations, molecular events and temporal sequelae in the inflammatory process. While remaining a burgeoning field in pre-clinical research, increasing application in man affords researchers the opportunity to study disease pathogenesis in humans in situ thereby revolutionizing conventional understanding of pathophysiology and potential therapeutic targets. This review provides a description of commonly used molecular imaging modalities, including optical, radionuclide and magnetic resonance imaging, and details key advances and translational opportunities in imaging inflammation from initiation to resolution.

Pain in neonates during screening for retinopathy of prematurity using binocular indirect ophthalmoscopy and wide-field digital retinal imaging: a randomised comparison.

OBJECTIVE: To compare the pain experienced by premature infants undergoing wide-field digital retinal imaging (WFDRI) and binocular indirect ophthalmoscopy (BIO) for retinopathy of prematurity (ROP) screening. METHODS: Infants were recruited at Edinburgh Royal Infirmary Neonatal Unit, Edinburgh, UK. Eyes were examined by WFDRI and BIO with eyelid speculum by two experienced paediatric ophthalmologists in random order. A pain score (Premature Infant Pain Profile (PIPP)) for WFDRI and BIO was generated. RESULTS: A total of 76 infants were recruited. The (mean, SD) PIPP score for WFDRI was 15.0, 2.1 and for BIO was 15.2, 2.4 (paired t test p=0.47). The authors observed that infants started crying with corresponding physiological changes as soon as the eyelid speculum was inserted and crying stopped on speculum removal. CONCLUSION: WFDRI and BIO with eyelid speculum are similarly painful for infants. The authors speculate that the eyelid speculum rather than the examination method may contribute most to the pain experienced.

The effects of chronic lead treatment and hypertension on the severity of cardiac arrhythmias induced by coronary artery occlusion or by noradrenaline in anaesthetised rats.

The aim of this study was to investigate whether chronic (3 months) lead (250 or 1000 ppm), administered as lead acetate in the drinking water, commencing either after weaning (in normotensive or spontaneously hypertensive male rats) or from conception (normotensive rats only) altered the susceptibility of the heart to arrhythmias induced either by coronary artery occlusion or by noradrenaline. Treatment with lead alone had no marked effect on the arrhythmias elicited by either method. Spontaneously hypertensive rats treated with either dose of lead exhibited more ectopic beats following coronary artery occlusion than normotensive rats but not more than those observed in control spontaneously hypertensive rats. An enhanced arrhythmogenic effect of noradrenaline was observed only in hypertensive rats administered 250 ppm lead. Both doses of lead accelerated the development of high blood pressure and in normotensive rats the higher dose also resulted in an elevated pressure. Following administration of lead, blood lead concentrations were elevated to 0.96 and 2.11 mumol l-1 after 250 and 1000 ppm, respectively. Accumulation of lead in heart and bone was also observed. We conclude that chronic exposure to these concentrations of lead, when combined with high blood pressure, slightly enhances the susceptibility of the heart to arrhythmias induced by myocardial ischaemia.

Liver tryptophan pyrrolase. A major determinant of the lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL mice.

The lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL, compared with -non-preferring CBA, mice is caused by a decrease in circulating tryptophan availability to the brain secondarily to a higher liver tryptophan pyrrolase activity associated with a higher circulating corticosterone concentration. Activity or expression of liver tryptophan pyrrolase and/or their induction by glucocorticoids may be important biological determinants of predisposition to alcohol consumption.

Stabilisation of Salmonella vaccine vectors by the induction of trehalose biosynthesis.

The growth of an aroA mutant of Salmonella typhimurium (SL3261) in minimal medium containing 0.5 M NaCl resulted in the intracellular accumulation of 2.2 micromol trehalose/mg total protein. The vacuum drying of these bacteria in the presence of trehalose allowed the recovery of 35% of the viable cells that were present before drying. In contrast, bacteria cultured in control medium accumulated 0.4 micromol trehalose/mg total protein and only 5% of the viable cells were recovered after vacuum drying with trehalose. Similar results were obtained when S. typhimurium SL3261, expressing the vaccine antigen (F1-antigen) of Yersinia pestis, was cultured in minimal medium with or without 0.5 M NaCl and dried in the presence of trehalose. Although these results indicate the potential for trehalose stabilisation of vaccine strains of S. typhimiurium, growth in minimal medium containing 0.5 M NaCl resulted in the loss of invasion competence of the bacteria.