Low-cost reversible tandem lens mesoscope for brain imaging in rodents.

Significance: The development of imaging systems that are cost-efficient and modular is essential for modern neuroscience research. Aim: In the current study, we designed, developed, and characterized a low-cost reversible tandem lens mesoscope for brain imaging in rodents. Approach: Using readily available components, we assembled a robust imaging system that is highly efficient and cost-effective. We developed a mesoscope that offers high-resolution structural and functional imaging with cost-effective lenses and CMOS camera. Results: The reversible tandem lens configuration of the mesoscope offers two fields of view (FOVs), which can be achieved by swapping the objective and imaging lenses. The large FOV configuration of 12.6×10.5 mm provides a spatial resolution up to 4.92 µm, and the small FOV configuration of 6×5 mm provides a resolution of up to 2.46 µm. We demonstrate the efficiency of our system for imaging neuronal calcium activity in both rat and mouse brains in vivo. Conclusions: The careful selection of the mesoscope components ensured its compactness, portability, and versatility, meaning that different types of samples and sample holders can be easily accommodated, enabling a range of different experiments both in vivo and in vitro. The custom-built reversible FOV mesoscope is cost-effective and was developed for under US$10,000 with excellent performance.

Rapid detection of viable microbes with 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride and 5(6)-carboxyfluorescein diacetate using a fibre fluorescence spectroscopy system.

AIMS: To assess the efficacy of two commercially available viability dyes, 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) and 5(6)-carboxyfluorescein diacetate (CFDA), in reporting on viable cell concentration and species using an all-fibre fluorometer. METHODS AND RESULTS: Four bacterial species (two Gram-positive and two Gram-negative) commonly associated with food poisoning or food spoilage (Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Bacillus cereus) were stained with CTC or CFDA and the fibre fluorometer was used to collect full fluorescence emission spectra. A good correlation between concentration and fluorescence intensity was found for Gram-negative bacteria between 107 and 108 colony-forming units (CFU) ml-1. There was no correlation with concentration for Gram-positive bacteria; however, the information in the CTC and CFDA spectra shows the potential to distinguish Gram-negative cells from Gram-positive cells, although it may simply reflect the overall bacterial metabolic activity under staining conditions from this study. CONCLUSIONS: The limit of detection (LoD) is too high in the dip-probe approach for analysis; however, the development of an approach measuring the fluorescence of single cells may improve this limitation. The development of new bacteria-specific fluorogenic dyes may also address this limitation. The ability to differentiate bacteria using these dyes may add value to measurements made to enumerate bacteria using CTC and CFDA.

Continuous separation of bacterial cells from large debris using a spiral microfluidic device.

With the global increase in food exchange, rapid identification and enumeration of bacteria has become crucial for protecting consumers from bacterial contamination. Efficient analysis requires the separation of target particles (e.g., bacterial cells) from food and/or sampling matrices to prevent matrix interference with the detection and analysis of target cells. However, studies on the separation of bacteria-sized particles and defined particles, such as bacterial cells, from heterogeneous debris, such as meat swab suspensions, are limited. In this study, we explore the use of passive-based inertial microfluidics to separate bacterial cells from debris, such as fascia, muscle tissues, and cotton fibers, extracted from ground meat and meat swabs-a novel approach demonstrated for the first time. Our objective is to evaluate the recovery efficiency of bacterial cells from large debris obtained from ground meat and meat swab suspensions using a spiral microfluidic device. In this study, we establish the optimal flow rates and Dean number for continuous bacterial cell and debris separation and a methodology to determine the percentage of debris removed from the sample suspension. Our findings demonstrate an average recovery efficiency of ∼80% for bacterial cells separated from debris in meat swab suspensions, while the average recovery efficiency from ground beef suspensions was ∼70%. Furthermore, approximately 50% of the debris in the ground meat suspension were separated from bacterial cells.

Broadband-excitation-based mechanical spectroscopy of highly viscous tissue-mimicking phantoms.

Standard rheometers assess mechanical properties of viscoelastic samples up to 100 Hz, which often hinders the assessment of the local-scale dynamics. We demonstrate that high-frequency analysis can be achieved by inducing broadband waves and monitoring their media-dependent propagation using optical coherence tomography. Here, we present a new broadband wave analysis based on two-dimensional Fourier transformation. We validated this method by comparing the mechanical parameters to monochromatic excitation and a standard oscillatory test data. Our method allows for high-frequency mechanical spectroscopy, which could be used to investigate the local-scale dynamics of different biological tissues and the influence of diseases on their microstructure.

Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents.

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when Escherichia coli was treated with lytic antibiotics-ampicillin and polymyxin B-and stained with SYTO 9 and/or propidium iodide. Antibiotic concentration, bacterial growth phase, and treatment time used affected the efficacy of this detection method. Quantification methods of the lethal action and inhibitory action of the non-lytic antibiotics, ciprofloxacin and chloramphenicol, respectively, remain to be elucidated.

The complex, bidirectional role of extracellular vesicles in infection.

Cells from all domains of life release extracellular vesicles (EVs), packages that carry a cargo of molecules that participate in communication, co-ordination of population behaviours, virulence and immune response mechanisms. Mammalian EVs play an increasingly recognised role to fight infection, yet may also be commandeered to disseminate pathogens and enhance infection. EVs released by bacterial pathogens may deliver toxins to host cells, signalling molecules and new DNA to other bacteria, and act as decoys, protecting infecting bacteria from immune killing. In this review, we explore the role of EVs in infection from the perspective of both the pathogen and host, and highlight their importance in the host/pathogen relationship. We highlight proposed strategies for EVs in therapeutics, and call attention to areas where existing knowledge and evidence is lacking.

A novel optical biosensor for in situ and small-scale monitoring of bacterial transport in saturated columns.

In situ monitoring techniques can provide new insight into bacterial transport after inoculating exogenous bacteria into contaminated soils for bioremediation. A real-time and non-destructive optical sensor (the optrode) was employed to monitor in situ transport of two fluorescently labelled bacteria - Green Fluorescent Protein (Gfp)-labelled, hydrophilic Pseudomonas putida and Tomato Fluorescent Protein (td)-labelled, hydrophobic Rhodococcus erythropolis, in a saturated sand column with and without rhamnolipid surfactant. In situ measurements were made at three sampling ports in the column with the optrode in two sets of column experiments. In Experiment 1, liquid samples were extracted for ex situ analyses (plate counts and fluorescence), while in Experiment 2 no liquid samples were extracted. Extracting liquid samples for ex situ analyses in Experiment 1 disturbed in situ measurements; in situ measured bacterial concentrations were lower, or a significant lag in breakthrough occurred relative to ex situ measurements. In Experiment 2, the optrode worked well in monitoring bacterial transport, which gave consistent transport parameters at each sampling port. Moreover, the optrode enabled the impact of bacterial hydrophobicity and rhamnolipid surfactant on bacterial transport to be observed. Specifically, hydrophilic P. putida was transported faster through the column than hydrophobic R. erythropolis; we infer from this result that fewer P. putida cells adsorb to sand particles than do R. erythropolis cells. The rhamnolipid surfactant enhanced the transport of both hydrophilic and hydrophobic bacteria. These two observations are consistent with Lifshitz-van der Waals forces and acid-base interactions between bacteria and sand.

Impact-induced cartilage damage assessed using polarisation-sensitive optical coherence tomography.

Non-invasive determination of structural changes in articular cartilage immediately after impact and rehydration provides insight into the response and recovery of the soft tissue, as well as provides a potential methodology for clinicians to quantify early degenerative changes. In this study, we use polarisation-sensitive optical coherence tomography (PS-OCT) to examine subtle alterations of the optical properties in healthy and early-stage degenerate articular cartilage immediately after impact loading to identify structurally relevant metrics required for understanding the mechanical factors of osteoarthritic initiation and progression. A custom-designed impact testing rig was used to deliver 0.9 J and 1.4 J impact energies to bovine articular cartilage. A total of 52 (n=26 healthy, n=26 mildly degenerate) cartilage-on-bone samples were imaged before, immediately after, and 3 h after impact. PS-OCT images were analyzed to assess changes relating to surface irregularity, optical attenuation, and birefringence. Mildly degenerate cartilage exhibits a significant change in birefringence following 1.4 J impact energies compared to healthy samples which is believed to be attributable to degenerate cartilage being unable to fully utilise the fluid phase to distribute and dampen the energy. After rehydration, the polarisation-sensitive images appear to 'optically-recover' reducing the reliability of birefringence as an absolute metric. Surface irregularity and optical attenuation encode diagnostically relevant information and may serve as markers to predict the mechanical response of articular cartilage. PS-OCT with its ability to non-invasively image the sub-surface microstructural abnormalities of cartilage presents as an ideal modality for cartilage degeneration assessment and identification of mechanically vulnerable tissue.

Towards real time assessment of intramuscular fat content in meat using optical fiber-based optical coherence tomography.

We consider the use of optical coherence tomography (OCT) imaging to predict the quality of meat. We find that intramuscular fat (IMF) absorbs infrared light about nine times stronger than muscle, which enables us to estimate fat content in intact meat samples. The method is made very efficient by extracting relevant information from the three-dimensional high-resolution images generated by OCT using principal component analysis (PCA). The principal components are then used as regressors into a support vector regression (SVR) prediction model. The SVR model is found to predict IMF content stably and accurately, with an R2 value of 0.94. Our study paves the way for automated, contact-less, non-destructive, real time classification of the quality of meat samples.

Species Dependence of SYTO 9 Staining of Bacteria.

SYTO 9 is a fluorescent nucleic acid stain that is widely used in microbiology, particularly for fluorescence microscopy and flow cytometry analyzes. Fluorimetry-based analysis, i.e., analysis of fluorescence intensity from a bulk sample measurement, is more cost effective, rapid and accessible than microscopy or flow cytometry but requires application-specific calibration. Here we show the relevance of SYTO 9 for food safety analysis. We stained four bacterial species of relevance to food safety (Bacillus cereus, Escherichia coli, Salmonella enterica subspecies enterica ser. Typhimurium, Staphylococcus aureus) with different concentrations of SYTO 9, with and without the presence of ethylenediaminetetraacetic acid (EDTA), for varying amounts of time, to investigate the effect of these treatment parameters on fluorescence intensity. The addition of EDTA and an increased staining duration did not significantly affect fluorescence intensity, and over the bacterial cell concentration range investigated (∼105-108 CFU/ml) there was no significant difference in using 0.5 or 1 µM SYTO 9. The effect of bacterial cell concentration on fluorescence intensity was species specific. At different bacterial cell concentrations, the effect of species on fluorescence intensity is different. This interaction complicates the development of a general fluorimetry-based protocol for the determination of bacterial cell concentration in a mixed bacterial suspension, as would be expected from samples taken from food safety settings.

Fourier domain quantum optical coherence tomography.

Quantum optical coherence tomography (Q-OCT) is the non-classical counterpart of optical coherence tomography (OCT), a high-resolution 3D imaging technique based on white-light interferometry. Because Q-OCT uses a source of frequency-entangled photon pairs, not only is the axial resolution not affected by dispersion mismatch in the interferometer but is also inherently improved by a factor of two. Unfortunately, practical applications of Q-OCT are hindered by image-scrambling artefacts and slow acquisition times. Here, we present a theoretical analysis of a novel approach that is free of these problems: Fourier domain Q-OCT (Fd-Q-OCT). Based on a photon pair coincidence detection as in the standard Q-OCT configuration, it also discerns each photon pair by their wavelength. We show that all the information about the internal structures of the object is encoded in the joint spectrum and can be easily retrieved through Fourier transformation. No depth scanning is required, making our technique potentially faster than standard Q-OCT. Finally, we show that the data available in the joint spectrum enables artefact removal and discuss prospective algorithms for doing so.

Augmentation of neural activity in peripheral nerve of sheep using 6 kHz subthreshold currents.

OBJECTIVE: To determine how increased excitability from subthreshold currents would alter neural activity as it propagates through the subthreshold currents. APPROACH: Experiments were performed on two Romney cross-breed sheep in vivo, by applying subthreshold currents either at the stimulus site or between the stimulus and recording sites. Neural recordings were obtained from nerve cuff implanted on the peroneal or sciatic nerve branches, while stimulus was applied to either the peroneal nerve or pins placed through the lower hindshank. MAIN RESULTS: Showed that subthreshold currents applied to the same site as stimulus increased excitation of underlying nerve fibres (p < 0.005). With stimulus and subthreshold currents applied to different sites on the peroneal nerve, the primary compound action potential (CAP) in the sciatic displayed a temporal shift of -2.5 to -3 µs which agreed with changes observed in the CAP waveform (p > 0.05). SIGNIFICANCE: These findings contribute to the understanding of mechanisms in myelinated fibres of subthreshold current neuromodulation therapies.

Asia-Pacific Optical Sensors Conference: focus issue introduction.

This feature issue of Optics Express contains 17 articles expanding on recent advances in optical sensors presented at the eighth Asia-Pacific Optical Sensors Conference (APOS 2019) held in Auckland, New Zealand, from November 19 to 22, 2019. These articles span sensing for real-time positioning, refractive indices, strain, gas, and temperature using a variety of methods including photoacoustic computed tomography, coherent optical frequency-modulated continuous-wave interferometry, enhanced Bragg gratings, and phase-sensitive optical frequency-domain reflectometry.

Quantum-inspired detection for spectral domain optical coherence tomography.

Intensity levels allowed by safety standards (ICNIRP or ANSI) limit the amount of light that can be used in a clinical setting to image highly scattering or absorptive tissues with optical coherence tomography (OCT). To achieve high-sensitivity imaging at low intensity levels, we adapt a detection scheme-which is used in quantum optics for providing information about spectral correlations of photons-into a standard spectral domain OCT system. This detection scheme is based on the concept of dispersive Fourier transformation, where a fiber introduces a wavelength-dependent time delay measured by a single-pixel detector, usually a high-speed photoreceiver. Here, we use a fast superconducting single-photon detector SSPD as a single-pixel detector and obtain images of a glass stack and a slice of onion at the intensity levels of the order of 10 pW. We also provide a formula for a depth-dependent sensitivity falloff in such a detection scheme, which can be treated as a temporal equivalent of diffraction-grating-based spectrometers.

Bourdieu, networks, and movements: Using the concepts of habitus, field and capital to understand a network analysis of gender differences in undergraduate physics.

Current trends suggest that significant gender disparities exist within Science, Technology, Engineering, and Mathematics (STEM) education at university, with female students being underrepresented in physics, but more equally represented in life sciences (e.g., biology, medicine). To understand these trends, it is important to consider the context in which students make decisions about which university courses to enrol in. The current study seeks to investigate gender differences in STEM through a unique approach that combines network analysis of student enrollment data with an interpretive lens based on the sociological theory of Pierre Bourdieu. We generate a network of courses taken by around 9000 undergraduate physics students (from 2009 to 2014) to quantify Bourdieu's concept of field. We identify the fields in which physics students participate by constructing a weighted co-enrollment network and finding communities within it. We then use odds ratios to report gender differences in transverse movements between different academic fields, and non-parametric tests to assess gender differences in vertical movements (changes in students' achievement rankings within a field). Odds ratios comparing the likelihood of progression from one field to another indicate that female students were more likely to make transverse movements into life science fields. We also found that university physics did a poor job in attracting high achieving students, and especially high achieving female students. Of the students who did choose to study physics at university, low and middle achieving female high school students were more likely to decrease their relative rank in their first year compared to their male counterparts. Low achieving female students were also less likely to continue with physics after their first year compared to their male counterparts. Results and implications are discussed in the context of Bourdieu's theory, and previous research. We argue that in order to remove constraints on female students' study choices, the field of physics needs to provide a culture in which all students feel like they belong.

Optical coherence tomography complements confocal microscopy for investigation of multicellular tumour spheroids.

Knowledge of optical properties, such as the refractive index (RI), of biological tissues is important in optical imaging, as they influence the distribution and propagation of light in tissue. To accurately study the response of cancerous cells to drugs, optimised imaging protocols are required. This study uses a simple custom-built spectral domain optical coherence tomography (OCT) system to conduct RI measurements of multicellular spheroids, three-dimensional (3D) in-vitro culture systems, of the cell line HCT116. The spheroid RIs are compared to study the effect of growth over time. To improve confocal microscopy imaging protocols, two immersion media (glycerol and ScaleView-A2) matching the spheroid RIs were trialled, with the aim to reduce the RI mismatch between the spheroid and the immersion medium and thus improve imaging depth with confocal microscopy. ScaleView-A2 (n = 1.380) aided in achieving greater depths of imaging of the multicellular spheroids under confocal microscopy. This improvement in imaging depth confirmed the utility of our RI measurements, proving the promising outlook of OCT as a complementary tool to microscopy in cancer research.

Rapid and cost-effective evaluation of bacterial viability using fluorescence spectroscopy.

A rapid and easy method that takes advantage of an inexpensive and portable fibre-based spectroscopic system (optrode) to determine the ratio of live to dead bacteria is proposed. Mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were stained using SYTO 9 and propidium iodide (PI) and measured using the optrode. We demonstrated several approaches to obtaining the proportions of live:dead E. coli in a mixture of both live and dead, from analyses of the fluorescence spectra collected by the optrode. To find a suitable technique for predicting the percentage of live bacteria in a sample, four analysis methods were assessed and compared: SYTO 9:PI fluorescence intensity ratio, an adjusted fluorescence intensity ratio, single-spectrum support vector regression (SVR) and multi-spectra SVR. Of the four analysis methods, multi-spectra SVR obtained the most reliable results and was able to predict the percentage of live bacteria in 108 bacteria/mL samples between c. 7 and 100% live, and in 107 bacteria/mL samples between c. 7 and 73% live. By demonstrating the use of multi-spectra SVR and the optrode to monitor E. coli viability, we raise points of consideration for spectroscopic analysis of SYTO 9 and PI and aim to lay the foundation for future work that uses similar methods for different bacterial species.

Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria.

Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should be completed to characterize limits of detection and range of linearity with respect to the plate count method. To demonstrate the calibration process, mixtures of Escherichia coli or Staphylococcus aureus with proportions of live and dead cells ranging from 0% to 100% were prepared. These samples were stained using nucleic acid-binding dyes, and 6 µm reference beads were added (LIVE/DEAD® BacLight kit). The calibration samples were analyzed using bead-based FCM as well as the agar plate count method, and the results from both methods were compared.

Optimisation of the Protocol for the LIVE/DEAD® BacLightTM Bacterial Viability Kit for Rapid Determination of Bacterial Load.

Rapid antimicrobial susceptibility testing is needed to reduce prescription of inappropriate antibiotics. A rapid alternative to standard culture-based testing is to determine reductions in cell viability using the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We optimised the kit protocol for this application, focusing on simplifying the process by minimising the steps involved and on determining the optimal analytical parameters for fluorescence measurements from the dyes SYTO 9 and propidium iodide (PI). We demonstrate that for our experimental system, the intensity of emissions should be integrated from 505-515 nm for SYTO 9 and 600-610 nm for PI, and the proportion of live cells calculated from a new dye ratio formula, termed the adjusted dye ratio. We show that the pre-staining washing step is not necessary if a non-fluorescent growth media is used; however, staining must be done for each sampling as prolonged exposure to the dyes negatively impacts cell viability. The optimised methodology was able to reproducibly detect reductions in culture viability when the proportion of live cells in a sample of 1 × 108 cells/ml fell below ∼50% live in a media that supports the growth required for detecting antibiotic killing. Finally, we show that the interaction of fluorescence emission spectra from SYTO 9 and PI stained Escherichia coli cells is influenced by the proportion of dead cells in a sample. The excitation of PI by SYTO 9 was found to occur in populations containing sufficient numbers of dead cells (>25%), whereas in populations with low numbers of dead cells the dye interaction was additive in regard to red emissions, indicating that these dye interactions may offer another dimension to live/dead analysis. Fluorescence measurements from samples established according to the optimised protocol can be taken using a flow cytometer, spectrofluorometer, microplate reader, and the Optrode, a fibre-based spectroscopic system developed at the University of Auckland.

Near real-time enumeration of live and dead bacteria using a fibre-based spectroscopic device.

A rapid, cost-effective and easy method that allows on-site determination of the concentration of live and dead bacterial cells using a fibre-based spectroscopic device (the optrode system) is proposed and demonstrated. Identification of live and dead bacteria was achieved by using the commercially available dyes SYTO 9 and propidium iodide, and fluorescence spectra were measured by the optrode. Three spectral processing methods were evaluated for their effectiveness in predicting the original bacterial concentration in the samples: principal components regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR). Without any sample pre-concentration, PCR achieved the most reliable results. It was able to quantify live bacteria from 108 down to 106.2 bacteria/mL and showed the potential to detect as low as 105.7 bacteria/mL. Meanwhile, enumeration of dead bacteria using PCR was achieved between 108 and 107 bacteria/mL. The general procedures described in this article can be applied or modified for the enumeration of bacteria within populations stained with fluorescent dyes. The optrode is a promising device for the enumeration of live and dead bacterial populations particularly where rapid, on-site measurement and analysis is required.