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.

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.

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.

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.

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.

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.

A rapid and low-cost estimation of bacteria counts in solution using fluorescence spectroscopy.

The fluorescence spectrum of bacterially bound acridine orange (AO) was investigated to evaluate its use for the rapid enumeration of bacteria. Escherichia coli ATCC 25922 samples were stained with 2 × 10-2, 2 × 10-3 or 2 × 10-4% w/v AO, followed by 3, 2 or 0 washing cycles, respectively, and fluorescence spectra were recorded using a fibre-based spectroscopic system. Independent component analysis was used to analyse the spectral datasets for each staining method. Bacterial concentration order of magnitude classification models were calculated using independent component weights. The relationship between fluorescence intensity of bound AO and bacterial concentration was not linear. However, the spectral signals collected for AO stain concentration-bacterial concentration pairs were reproducible and unique enough to enable classification of samples. When above 105 CFU ml-1, it was possible to rapidly determine what the order of magnitude of bacterial concentration of a sample was using a combination of two of the sample preparation methods. A relatively inexpensive (around US$10 per test) rapid method (within 25 min of sampling) for enumeration of bacteria by order of magnitude will reduce the time and cost of microbiological tests requiring gross concentration information. Graphical Abstract Fluorescence spectra of bacterially bound acridine orange (AO) were used for the rapid enumeration of bacteria. Order of magnitude bacterial concentration classification models were calculated using independent components analysis of these fluorescence spectra. When above 105 CFU ml-1, it was possible to rapidly determine the order of magnitude of bacterial concentration of a sample using a combination of two sample preparation methods.

Dispersion measurements in ocular media using a dual-wavelength swept source optical coherence tomography system.

Optical coherence tomography (OCT) has proved to be a powerful tool for the detection of microstructure in tissue. Label-free tissue differentiation on a micron scale is a promising and powerful technique for segmentation. This Letter describes a technique using a dual-wavelength OCT system to image the eye. We measure the walk-off between interfaces in A-scans, taken at two different wavelengths, to calculate the average group velocity dispersion parameter of each segment of the eye. We present measurements of the dispersion of the cornea and the aqueous humour in rat eyes.

3D super-resolved in vitro multiphoton microscopy by saturation of excitation.

We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.

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.

Single-shot speckle reduction and dispersion compensation in optical coherence tomography by compounding fractional Fourier domains.

We present speckle suppression and dispersion compensation for Fourier-domain optical coherence tomography based on fractional Fourier transforms of a single A scan. A 1.54-fold reduction in speckle contrast was achieved with group velocity dispersion compensation. The method is demonstrated on biological samples using a swept source configuration at 1310 nm and a spectral-domain system at 840 nm.

Dispersion mapping at the micrometer scale using tri-band optical frequency domain imaging.

Techniques to differentiate between materials are a powerful addition to the structural information traditionally available from optical coherence tomography images. We present label-free detection of water and lipid at a micrometer scale by evaluating their unique dispersion properties. Using a tri-band swept source configuration, we measure both ß(2) and ß(3) and show how to identify the two materials at sample thicknesses of 40 and 90 µm, respectively.

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.

Dispersion compensation in Fourier domain optical coherence tomography using the fractional Fourier transform.

We address numerical dispersion compensation based on the use of the fractional Fourier transform (FrFT). The FrFT provides a new fundamental perspective on the nature and role of group-velocity dispersion in Fourier domain OCT. The dispersion induced by a 26 mm long water cell was compensated for a spectral bandwidth of 110 nm, allowing the theoretical axial resolution in air of 3.6 µm to be recovered from the dispersion degraded point spread function. Additionally, we present a new approach for depth dependent dispersion compensation based on numerical simulations. Finally, we show how the optimized fractional Fourier transform order parameter can be used to extract the group velocity dispersion coefficient of a material.

Instantaneous quadrature components or Jones vector retrieval using the Pancharatnam-Berry phase in frequency domain low-coherence interferometry.

We use the Pancharatnam-Berry phase as a multifunctional tool for low-coherence interferometry. This geometric phase shift enables instantaneous retrieval of the quadrature components of the complex interferometric signal. The phase shift is independent of wavelength and allows for a complex conjugate suppression of 40 dB for an optical bandwidth of 115 nm. Furthermore, this paper investigates the versatility of the geometric phase to perform polarization sensitive measurements. The Jones vector of the sample was obtained numerically, allowing sample birefringence and optical axis calculation.

Novel fiber optic detection method for in situ analysis of fluorescently labeled biosensor organisms.

This research presents a novel, time-resolved fiber-optic "Optrode" system for accurate real-time in situ detection of fluorescent proteins produced by biosensor organisms. The Optrode fluorescence detection system was able to identify, characterize, differentiate, and quantify red and green fluorescently labeled organisms, individually and in mixed aqueous cultures. Detection was also possible in sand systems, where a consistent reduction in signal intensity indicates that signal collection volume was reduced by one-third. The optrode was shown to be sensitive enough to detect fluorescently labeled cell concentrations of 1.9 × 10(4) CFU/mL, indicating it is suitable for detecting typical concentrations of degrader organisms reported in bioremediation trials. The effect of fluorophore photobleaching was characterized for different fluorescent proteins and demonstrated a linear relationship to cell concentration, meaning the effect can be accounted for within methods of fluorescence collection and analysis. Proof of concept is provided for all aspects of this research, which represents an important step toward the goal of achieving a complete, nondestructive, in situ monitoring system to characterize all aspects of microbial activity and gene expression.

Improving the efficiency of optical coherence tomography by using the non-ideal behaviour of a polarising beam splitter.

We present a new way of improving the efficiency of optical coherence tomography by using the polarisation crosstalk of a polarising beam splitter to direct most of the available source optical power to the sample. The use of a quarter wave plate in both the reference and the sample arms allows most of the sample power to be directed to the detector while adjusting the reference arm to ensure noise optimised operation. As a result, the sensitivity of such a system can be improved by 6 dB, or alternatively the acquisition time can be improved by a factor of 4 for shot noise limited performance,compared to a traditional OCT configuration using a 50/50 beam splitter.

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.