Sensing the Spin State of Room-Temperature Switchable Cyanometallate Frameworks with Nitrogen-Vacancy Centers in Nanodiamonds.

Room-temperature magnetically switchable materials play a vital role in current and upcoming quantum technologies, such as spintronics, molecular switches, and data storage devices. The increasing miniaturization of device architectures produces a need to develop analytical tools capable of precisely probing spin information at the single-particle level. In this work, we demonstrate a methodology using negatively charged nitrogen vacancies (NV-) in fluorescent nanodiamond (FND) particles to probe the magnetic switching of a spin crossover (SCO) metal-organic framework (MOF), [Fe(1,6-naphthyridine)2(Ag(CN)2)2] material (1), and a single-molecule photomagnet [X(18-crown-6)(H2O)3]Fe(CN)6·2H2O, where X = Eu and Dy (materials 2a and 2b, respectively), in response to heat, light, and electron beam exposure. We employ correlative light-electron microscopy using transmission electron microscopy (TEM) finder grids to accurately image and sense spin-spin interacting particles down to the single-particle level. We used surface-sensitive optically detected magnetic resonance (ODMR) and magnetic modulation (MM) of FND photoluminescence (PL) to sense spins to a distance of ca. 10-30 nm. We show that ODMR and MM sensing was not sensitive to the temperature-induced SCO of FeII in 1 as formation of paramagnetic FeIII through surface oxidation (detected by X-ray photoelectron spectroscopy) on heating obscured the signal of bulk SCO switching. We found that proximal FNDs could effectively sense the chemical transformations induced by the 200 keV electron beam in 1, namely, AgI → Ag0 and FeII → FeIII. However, transformations induced by the electron beam are irreversible as they substantially disrupt the structure of MOF particles. Finally, we demonstrate NV- sensing of reversible photomagnetic switching, FeIII + (18-crown-6) ⇆ FeII + (18-crown-6)+ •, triggered in 2a and 2b by 405 nm light. The photoredox process of 2a and 2b proved to be the best candidate for room-temperature single-particle magnetic switching utilizing FNDs as a sensor, which could have applications into next-generation quantum technologies.

Nitrogen vacancy defects in single-particle nanodiamonds sense paramagnetic transition metal spin noise from nanoparticles on a transmission electron microscopy grid.

Spin-active nanomaterials play a vital role in current and upcoming quantum technologies, such as spintronics, data storage and computing. To advance the design and application of these materials, methods to link size, shape, structure, and chemical composition with functional magnetic properties at the nanoscale level are needed. In this work, we combine the power of two local probes, namely, Nitrogen Vacancy (NV) spin-active defects in diamond and an electron beam, within experimental platforms used in electron microscopy. Negatively charged NVs within fluorescent nanodiamond (FND) particles are used to sense the local paramagnetic environment of Rb0.5Co1.3[Fe(CN)6]·3.7H2O nanoparticles (NPs), a Prussian blue analogue (PBA), as a function of FND-PBA distance (order of 10 nm) and local PBA concentration. We demonstrate perturbation of NV spins by proximal electron spins of transition metals within NPs, as detected by changes in the photoluminescence (PL) of NVs. Workflows are reported and demonstrated that employ a Transmission Electron Microscope (TEM) finder grid to spatially correlate functional and structural features of the same unique NP studied using NV sensing, based on a combination of Optically Detected Magnetic Resonance (ODMR) and Magnetic Modulation (MM) of NV PL, within TEM imaging modalities. Significantly, spin-spin dipole interactions were detected between NVs in a single FND and paramagnetic metal centre spin fluctuations in NPs through a carbon film barrier of 13 nm thickness, evidenced by TEM tilt series imaging and Electron Energy-Loss Spectroscopy (EELS), opening new avenues to sense magnetic materials encapsulated in or between thin-layered nanostructures. The measurement strategies reported herein provide a pathway towards solid-state quantitative NV sensing with atomic-scale theoretical spatial resolution, critical to the development of quantum technologies, such as memory storage and molecular switching nanodevices.

Preliminary demonstration of benchtop NMR metabolic profiling of feline urine: chronic kidney disease as a case study.

OBJECTIVE: The use of benchtop metabolic profiling technology based on nuclear magnetic resonance (NMR) was evaluated in a small cohort of cats with a view to applying this as a viable and rapid metabolic tool to support clinical decision making. RESULTS: Urinary metabolites were analysed from four subjects consisting of two healthy controls and two chronic kidney disease (CKD) IRIS stage 2 cases. The study identified 15 metabolites in cats with CKD that were different from the controls. Among them were acetate, creatinine, citrate, taurine, glycine, serine and threonine. Benchtop NMR technology is capable of distinguishing between chronic kidney disease case and control samples in a pilot feline cohort based on metabolic profile. We offer perspectives on the further development of this pilot work and the potential of the technology, when combined with sample databases and computational intelligence techniques to offer a clinical decision support tool not only for cases of renal disease but other metabolic conditions in the future.

Progress in low-field benchtop NMR spectroscopy in chemical and biochemical analysis.

The employment of spectroscopically-resolved NMR techniques as analytical probes have previously been both prohibitively expensive and logistically challenging in view of the large sizes of high-field facilities. However, with recent advances in the miniaturisation of magnetic resonance technology, low-field, cryogen-free "benchtop" NMR instruments are seeing wider use. Indeed, these miniaturised spectrometers are utilised in areas ranging from food and agricultural analyses, through to human biofluid assays and disease monitoring. Therefore, it is both intrinsically timely and important to highlight current applications of this analytical strategy, and also provide an outlook for the future, where this approach may be applied to a wider range of analytical problems, both qualitatively and quantitatively.

Quantum Sensing in a Physiological-Like Cell Niche Using Fluorescent Nanodiamonds Embedded in Electrospun Polymer Nanofibers.

Fluorescent nanodiamonds (fNDs) containing nitrogen vacancy (NV) centers are promising candidates for quantum sensing in biological environments. This work describes the fabrication and implementation of electrospun poly lactic-co-glycolic acid (PLGA) nanofibers embedded with fNDs for optical quantum sensing in an environment, which recapitulates the nanoscale architecture and topography of the cell niche. A protocol that produces uniformly dispersed fNDs within electrospun nanofibers is demonstrated and the resulting fibers are characterized using fluorescent microscopy and scanning electron microscopy (SEM). Optically detected magnetic resonance (ODMR) and longitudinal spin relaxometry results for fNDs and embedded fNDs are compared. A new approach for fast detection of time varying magnetic fields external to the fND embedded nanofibers is demonstrated. ODMR spectra are successfully acquired from a culture of live differentiated neural stem cells functioning as a connected neural network grown on fND embedded nanofibers. This work advances the current state of the art in quantum sensing by providing a versatile sensing platform that can be tailored to produce physiological-like cell niches to replicate biologically relevant growth environments and fast measurement protocols for the detection of co-ordinated endogenous signals from clinically relevant populations of electrically active neuronal circuits.

Low-Field, Benchtop NMR Spectroscopy as a Potential Tool for Point-of-Care Diagnostics of Metabolic Conditions: Validation, Protocols and Computational Models.

Novel sensing technologies for liquid biopsies offer promising prospects for the early detection of metabolic conditions through omics techniques. Indeed, high-field nuclear magnetic resonance (NMR) facilities are routinely used for metabolomics investigations on a range of biofluids in order to rapidly recognise unusual metabolic patterns in patients suffering from a range of diseases. However, these techniques are restricted by the prohibitively large size and cost of such facilities, suggesting a possible role for smaller, low-field NMR instruments in biofluid analysis. Herein we describe selected biomolecule validation on a low-field benchtop NMR spectrometer (60 MHz), and present an associated protocol for the analysis of biofluids on compact NMR instruments. We successfully detect common markers of diabetic control at low-to-medium concentrations through optimised experiments, including α-glucose (≤2.8 mmol/L) and acetone (25 µmol/L), and additionally in readily accessible biofluids, particularly human urine. We present a combined protocol for the analysis of these biofluids with low-field NMR spectrometers for metabolomics applications, and offer a perspective on the future of this technique appealing to 'point-of-care' applications.

Ultrasound-mediation of self-illuminating reporters improves imaging resolution in optically scattering media.

In vivo imaging of self-illuminating bio-and chemiluminescent reporters is used to observe the physiology of small animals. However, strong light scattering by biological tissues results in poor spatial resolution of the optical imaging, which also degrades the quantitative accuracy. To overcome this challenging problem, focused ultrasound is used to modulate the light from the reporter at the ultrasound frequency. This produces an ultrasound switchable light 'beacon' that reduces the influence of light scattering in order to improve spatial resolution. The experimental results demonstrate that apart from light modulation at the ultrasound frequency (AC signal at 3.5 MHz), ultrasound also increases the DC intensity of the reporters. This is shown to be due to a temperature rise caused by insonification that was minimized to be within acceptable mammalian tissue safety thresholds by adjusting the duty cycle of the ultrasound. Line scans of bio-and chemiluminescent objects embedded within a scattering medium were obtained using ultrasound modulated (AC) and ultrasound enhanced (DC) signals. Lateral resolution is improved by a factor of 12 and 7 respectively, as compared to conventional CCD imaging. Two chemiluminescent sources separated by ~10 mm at ~20 mm deep inside a 50 mm thick chicken breast have been successfully resolved with an average signal-to-noise ratio of approximately 8-10 dB.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

Nanoscale Ultrasound-Switchable FRET-Based Liposomes for Near-Infrared Fluorescence Imaging in Optically Turbid Media.

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound-switchable FRET-based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near-infrared window are prepared. The efficacy of FRET and self-quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound-switchable FRET-based liposomes studied here have for in vivo fluorescence imaging.

Ultrasound Induced Fluorescence of Nanoscale Liposome Contrast Agents.

A new imaging contrast agent is reported that provides an increased fluorescent signal upon application of ultrasound (US). Liposomes containing lipids labelled with pyrene were optically excited and the excimer fluorescence emission intensity was detected in the absence and presence of an ultrasound field using an acousto-fluorescence setup. The acousto-fluorescence dynamics of liposomes containing lipids with pyrene labelled on the fatty acid tail group (PyPC) and the head group (PyPE) were compared. An increase in excimer emission intensity following exposure to US was observed for both cases studied. The increased intensity and time constants were found to be different for the PyPC and PyPE systems, and dependent on the applied US pressure and exposure time. The greatest change in fluorescence intensity (130%) and smallest rise time constant (0.33 s) are achieved through the use of PyPC labelled liposomes. The mechanism underlying the observed increase of the excimer emission intensity in PyPC labelled liposomes is proposed to arise from the "wagging" of acyl chains which involves fast response and requires lower US pressure. This is accompanied by increased lipid lateral diffusivity at higher ultrasound pressures, a mechanism that is also active in the PyPE labelled liposomes.

The interactions of squalene, alkanes and other mineral oils with model membranes; effects on membrane heterogeneity and function.

Droplet interface bilayers (DIBs) offer many favourable facets as an artificial membrane system but the influence of any residual oil that remains in the bilayer following preparation is ill-defined. In this study the fluorescent membrane probes di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (Di-8-ANEPPS) and Fluoresceinphosphatidylethanolamine (FPE) were used to help understand the nature of the phospholipid-oil interaction and to examine any structural and functional consequences of such interactions on membrane bilayer properties. Concentration-dependent modifications of the membrane dipole potential were found to occur in phospholipid vesicles exposed to a variety of different oils. Incorporation of oil into the lipid bilayer was shown to have no significant effect on the movement of fatty acids across the lipid bilayer. Changes in membrane heterogeneity were, however, demonstrated with increased microdomain formation being visible in the bilayer following exposure to mineral oil, pentadecane and squalene. As it is important that artificial systems provide an accurate representation of the membrane environment, careful consideration should be taken prior to the application of DIBs in studies of membrane structure and organisation.

Ultrasound modulated optical tomography contrast enhancement with non-linear oscillation of microbubbles.

BACKGROUND: Ultrasound modulated optical tomography (USMOT) is an imaging technique used to provide optical functional information inside highly scattering biological tissue. One of the challenges facing this technique is the low image contrast. METHODS: A contrast enhancement imaging technique based on the non-linear oscillation of microbubbles is demonstrated to improve image contrast. The ultrasound modulated signal was detected using a laser pulse based speckle contrast detection system. Better understanding of the effects of microbubbles on the optical signals was achieved through simultaneous measurement of the ultrasound scattered by the microbubbles. RESULTS: The length of the laser pulse was found to affect the system response of the speckle contrast method with shorter pulses suppressing the fundamental ultrasound modulated optical signal. Using this property, image contrast can be enhanced by detection of the higher harmonic ultrasound modulated optical signals due to nonlinear oscillation and destruction of the microbubbles. Experimental investigations were carried out to demonstrate a doubling in contrast by imaging a scattering phantom containing an embedded silicone tube with microbubbles flowing through it. CONCLUSIONS: The contrast enhancement in USMOT resulting from the use of ultrasound microbubbles has been demonstrated. Destruction of the microbubbles was shown to be the dominant effect leading to contrast improvement as shown by simultaneously detecting the ultrasound and speckle contrast signals. Line scans of a microbubble filled silicone tube embedded in a scattering phantom demonstrated experimentally the significant image contrast improvement that can be achieved using microbubbles and demonstrates the potential as a future clinical imaging tool.

Numerical Investigation of the Mechanisms of Ultrasound-Modulated Bioluminescence Tomography.

OBJECTIVE: A hybrid imaging technique, ultrasound-modulated luminescence tomography, that uses ultrasound to modulate diffusely propagating light has been shown to improve the spatial resolution of optical images. This paper investigates the underlying modulation mechanisms and the feasibility of applying this technique to improve spatial resolution in bioluminescence tomography. METHODS: Ultrasound-modulated bioluminescence tomography was studied numerically to identify the effects of four factors (reduced optical scattering coefficient, optical absorption coefficient, refractive index, and luciferase concentration) on the depth of light modulation. In practice, an open source finite-element method tool for simulation of diffusely propagating light, near infrared fluorescence and spectral tomography, was modified to incorporate the effects of ultrasound modulation. The signal-to-noise ratios of detected modulated bioluminescent emissions are calculated using the optical and physical properties of a mouse model. RESULTS: The modulation depth of the bioluminescent emission affected by the US induced variation of local concentration of the light emitting enzyme luciferase was at least two orders of magnitude greater than that caused by variations in the other factors. For surface radiances above approximately 10(7) photons/s/cm(2)/sr, the corresponding SNRs are detectable with the currently available detector technologies. CONCLUSION: The dominant effect in generation of ultrasound-modulated bioluminescence is ultrasound induced variation in luciferase concentration. The SNR analysis confirms the feasibility of applying ultrasound-modulated bioluminescence tomography in preclinical imaging of mice. SIGNIFICANCE: The simulation model developed suggests ultrasound-modulated bioluminescence tomography is a potential technique to improve the spatial resolution of bioluminescence tomography.

Ultrasonic monitoring of drug loaded Pluronic F127 micellular hydrogel phase behaviour.

Pluronic hydrogels composed of PEO-PPO-PEO tri-block copolymers have received a lot of attention for their applicability to drug delivery. These systems can be injected into the body in a liquid form and then, in response to temperature changes, self-assemble into nano-sized micelles which ultimately aggregate to form a gel. The phase behaviour and effectiveness of Pluronic hydrogels as drug carriers is affected by the local thermal and ionic environment which is likely to be different from patient to patient. There is a current need for in vivo techniques to study the phase behaviour of Pluronic hydrogels and this work demonstrates an ultrasound approach for the study of drug loaded Pluronic F127 hydrogels. Ultrasound velocity and attenuation were both found to change with temperature and through validation with fluorescence spectroscopy it was determined that the temperature dependent micellation transition in the Pluronic solutions could be identified through relative changes in ultrasound velocity and attenuation as a function of temperature. This phase transition was more clearly detected through examination of the first and second derivatives of both ultrasound parameters with respect to temperature. Further this work demonstrates for the first time to our knowledge ultrasound characterisation studies on drug loaded Pluronics.

Pulsed ultrasound modulated optical tomography with harmonic lock-in holography detection.

A method that uses digital heterodyne holography reconstruction to extract scattered light modulated by a single-cycle ultrasound (US) burst is demonstrated and analyzed. An US burst is used to shift the pulsed laser frequency by a series of discrete harmonic frequencies which are then locked on a CCD. The analysis demonstrates that the unmodulated light's contribution to the detected signal can be canceled by appropriate selection of the pulse repetition frequency. It is also shown that the modulated signal can be maximized by selecting a pulse sequence which consists of a pulse followed by its inverted counterpart. The system is used to image a 12 mm thick chicken breast with 2 mm wide optically absorbing objects embedded at the midplane. Furthermore, the method can be revised to detect the nonlinear US modulated signal by locking at the second harmonic US frequency.

Tissue transglutaminase (TG-2) modified amniotic membrane: a novel scaffold for biomedical applications.

The amniotic membrane (AM) is considered as a natural cell culture substrate and has occasionally been exploited in regenerative medicine especially for ocular surface reconstruction and dermal wound healing applications. However, its use is limited by its relatively weak mechanical strength, difficulty during manual handling and susceptibility to proteolytic degradation in vivo. Therefore, in this study we aimed to enhance the mechanical and biological characteristics of the AM by enzymatically cross-linking it using tissue transglutaminase (TG)-a calcium-dependent enzyme capable of forming stable ε(γ-glutamyl)lysine cross-linkages. Using a biological catalyst such as TG does not only prevent denaturation during sample preparation but also minimizes the potential of residual chemical cross-linking agents compared to alternative methodologies. Human AM, sourced from elective caesarean sectioning, were treated with TG, bovine serum albumin and/or a no-treatment control. Samples were then compared in terms of their physical and (scanning electron microscopy (SEM), transparency, mechanical strength, susceptibility to proteolytic degradation) biological characteristics (in vitro cell culture, activation of dendritic cells (DC)) and their in vivo biocompatibility/angiogenic capacity (chick chorioallantoic membrane assay). TG-treated AM exhibited enhanced mechanical strength and greater resistance to proteolytic/collagenase degradation compared to the control(s). SEM imaging of the TG-treated membrane summarized a significantly closer association and greater interconnectivity of individual collagen fibres yet it had no effect on the overall transparency of the AM. In vitro cell culture demonstrated no detrimental effect of TG-treatment on the AM in terms of cell attachment, spreading, proliferation and differentiation. Moreover, an 'immune response' was not elicited based on extended in vitro culture with human-monocyte-derived DC. Interestingly, the TG-treated AM still allowed angiogenesis to occur and in some instances, demonstrated an enhancement compared to the control (n = 5). We hereby demonstrate that treating the AM with the cross-linking enzyme, TG, results in a novel biomaterial with enhanced mechanical and biological characteristics. Above all, this modified membrane demonstrates greater strength, maintains in vitro cell growth, retains optical transparency and allows angiogenesis to occur without inducing an immune response. Altogether, this study demonstrates the feasibility of TG as an alternate cross-linking treatment for the production of novel biomaterials and suggests that TG-treated AM may now be more commonly exploited as a therapeutic dressing for ocular or wound applications.

Pulse inversion ultrasound modulated optical tomography.

Pulse inversion acoustic imaging is useful as it allows second harmonic imaging to be obtained with short acoustic pulses. This allows high axial resolution, but removes any overlap in the frequency spectra of fundamental and harmonic. We demonstrate pulse inversion ultrasound modulated optical tomography using an optical speckle based detection method. Inverted and non-inverted acoustic pulses combined with synchronized strobed illumination are applied to an optically scattering medium. Over the acquisition time of a camera, multiple pulses are summed and at the next frame the phase of the ultrasound is shifted by π/2 and the process repeated. Combining the two frames allows a second harmonic signal to be obtained. A reduction in linewidth is observed (DC=9.26 mm, fundamental=4.02 mm, second harmonic=2.43 mm) in line scans of optically absorbing objects embedded in a scattering medium (thickness=16 mm, scattering coefficient=2.3 mm(-1), anisotropy factor=0.938).

Application of a maximum likelihood algorithm to ultrasound modulated optical tomography.

In pulsed ultrasound modulated optical tomography (USMOT), an ultrasound (US) pulse performs as a scanning probe within the sample as it propagates, modulating the scattered light spatially distributed along its propagation axis. Detecting and processing the modulated signal can provide a 1-dimensional image along the US axis. A simple model is developed wherein the detected signal is modelled as a convolution of the US pulse and the properties (ultrasonic/optical) of the medium along the US axis. Based upon this model, a maximum likelihood (ML) method for image reconstruction is established. For the first time to our knowledge, the ML technique for an USMOT signal is investigated both theoretically and experimentally. The ML method inverts the data to retrieve the spatially varying properties of the sample along the US axis, and a signal proportional to the optical properties can be acquired. Simulated results show that the ML method can serve as a useful reconstruction tool for a pulsed USMOT signal even when the signal-to-noise ratio (SNR) is close to unity. Experimental data using 5 cm thick tissue phantoms (scattering coefficient µ(s) = 6.5 cm(-1), anisotropy factor g=0.93) demonstrate that the axial resolution is 160 µm and the lateral resolution is 600 µm using a 10 MHz transducer.

Time-optimized X-ray micro-CT imaging of polymer based scaffolds.

Although X-ray microscopic computed tomography is widely used to assess the structural properties of polymeric tissue scaffolds its validity is dependent on the quality of the images obtained. Here, the role of resolution, integration time, image averaging, and X-ray power on the accurate determination of scaffold porosity, while aiming to minimize imaging time, was investigated. This work identified key parameters for optimization and a methodology to vary them to improve results. Based on this, guidelines were developed to assist in the selection of image acquisition parameters to allow rapid and accurate scaffold imaging as required for mass manufacture.

Hydrogels in regenerative medicine: towards understanding structure-function relationships.

Hydrogels are playing an increasing role in regenerative medicine owing to their growing functional sophistication. This is being underpinned by advances in hydrogel synthesis, particularly through molecular and genetic engineering, which provide greater control of hydrogel structure and hence the emergence of hydrogels with new functionalities. In order to exploit this capability it is necessary to fully understand the relationship between hydrogel structure and function. This article will investigate the nature of hydrogel-structure relationships by: highlighting the key attributes of hydrogels that modulate their function, discussing the link between these attributes and hydrogel behavior, and identifying possible measurement strategies to elucidate them.