Monte Carlo simulations and phantom modeling for spatial frequency domain imaging of surgical wound monitoring.

Significance: Postoperative surgical wound infection is a serious problem around the globe, including in countries with advanced healthcare systems, and a method for early detection of infection is urgently required. Aim: We explore spatial frequency domain imaging (SFDI) for distinguishing changes in surgical wound healing based on the tissue scattering properties and surgical wound width measurements. Approach: A comprehensive numerical method is developed by applying a three-dimensional Monte Carlo simulation to a vertical heterogeneous wound model. The Monte Carlo simulation results are validated using resin phantom imaging experiments. Results: We report on the SFDI lateral resolution with varying reduced scattering value and wound width and discuss the partial volume effect at the sharp vertical boundaries present in a surgical incision. The detection sensitivity of this method is dependent on spatial frequency, wound reduced scattering coefficient, and wound width. Conclusions: We provide guidelines for future SFDI instrument design and explanation for the expected error in SFDI measurements.

Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production.

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10-15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

Evaluation of cataract formation in fish exposed to environmental radiation at Chernobyl and Fukushima.

Recent studies apparently finding deleterious effects of radiation exposure on cataract formation in birds and voles living near Chernobyl represent a major challenge to current radiation protection regulations. This study conducted an integrated assessment of radiation exposure on cataractogenesis using the most advanced technologies available to assess the cataract status of lenses extracted from fish caught at both Chernobyl in Ukraine and Fukushima in Japan. It was hypothesised that these novel data would reveal positive correlations between radiation dose and early indicators of cataract formation. The structure, function and optical properties of lenses were analysed from atomic to millimetre length scales. We measured the short-range order of the lens crystallin proteins using Small Angle X-Ray Scattering (SAXS) at both the SPring-8 and DIAMOND synchrotrons, the profile of the graded refractive index generated by these proteins, the epithelial cell density and organisation and finally the focal length of each lens. The results showed no evidence of a difference between the focal length, the epithelial cell densities, the refractive indices, the interference functions and the short-range order of crystallin proteins (X-ray diffraction patterns) in lens from fish exposed to different radiation doses. It could be argued that animals in the natural environment which developed cataract would be more likely, for example, to suffer predation leading to survivor bias. But the cross-length scale study presented here, by evaluating small scale molecular and cellular changes in the lens (pre-cataract formation) significantly mitigates against this issue.

Integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom spectrometers.

We present an integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom hyperspectral imagers. The system comprises of a cosine corrector and custom fiber patch cables, collecting the ambient light in a large solid angle and feeding it directly to the entrance slit of the spectrometer. The system enables simultaneous measurement of downwelling and upwelling irradiance using the main hyperspectral camera sensor. As a demonstration, the spectral reflectance of a soil sample was measured with a RMSE of 8.4%, a significant improvement on the RMSE of 54% found without correction. At a weight of approximately 10 grams, this system provides a substantial weight saving over standalone incident light sensing instruments.

Spectral index selection method for remote moisture sensing under challenging illumination conditions.

Remote sensing using passive solar illumination in the Short-Wave Infrared spectrum is exposed to strong intensity variation in the spectral bands due to atmospheric changing conditions and spectral absorption. More robust spectral analysis methods, insensitive to these effects, are increasingly required to improve the accuracy of the data analysis in the field and extend the use of the system to "non ideal" illumination condition. A computational hyperspectral image analysis method (named HIAM) for deriving optimal reflectance indices for use in remote sensing of soil moisture content is detailed and demonstrated. Using histogram analysis of hyperspectral images of wet and dry soil, contrast ratios and wavelength pairings were tested to find a suitable spectral index to recover soil moisture content. Measurements of local soil samples under laboratory and field conditions have been used to demonstrate the robustness of the index to varying lighting conditions, while publicly available databases have been used to test across a selection of soil classes. In both cases, the moisture was recovered with RMS error better than 5%. As the method is independent of material type, this method has the potential to also be applied across a variety of biological and man-made samples.

Full spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution.

The use of optical techniques to interrogate wide ranging samples from semiconductors to biological tissue for rapid analysis and diagnostics has gained wide adoption over the past decades. The desire to collect ever more spatially, spectrally and temporally detailed optical signatures for sample characterization has specifically driven a sharp rise in new optical microscopy technologies. Here we present a high-speed optical scanning microscope capable of capturing time resolved images across 512 spectral and 32 time channels in a single acquisition with the potential for ~0.2 frames per second (256 × 256 image pixels). Each pixel in the resulting images contains a detailed data cube for the study of diverse time resolved light driven phenomena. This is enabled by integration of system control electronics and on-chip processing which overcomes the challenges presented by high data volume and low imaging speed, often bottlenecks in previous systems.

Freeform based hYperspectral imager for MOisture Sensing (FYMOS).

We present FYMOS, an all-aluminum, robust, light weight, freeform based, near infrared hYperspectral imager for MOisture Sensing. FYMOS was designed and built to remotely measure moisture content using spectral features from 0.7-1.7µm integrating an InGaAs sensor. The imaging system, operating at F/2.8, is based on the three-concentric-mirror (Offner) spectrograph configuration providing a spectral resolution of 8 nm optimized for broad spectral coverage with sufficient resolution to make assessments of water levels. To optimize the optical performance, whilst minimizing weight and size, the design incorporates a bespoke freeform blazed grating machined on a commercial 5 axis ultra precision diamond machine. We achieve a 30% improvement on the RMS wavefront error in the spatial and spectral fields compared to a conventional Offner-Chrisp design with similar aperture and the monolithic Primary/Tertiary mirror eases the manufacturing assembly whilst minimizing weight. We demonstrate the performance of FYMOS by measuring the evaporation rate of water on a soil sample and results are processed with a physical multilayer radiative transfer model (MARMIT) to estimate the mean water thickness.

Feature issue introduction: applications of adaptive optics.

This feature issue of Optics Express follows the 2020 Imaging and Applied Optics Congress and comprises of articles on the development and use of adaptive optics across the broad range of domains in which the technique has been applied - including atmospheric correction, ophthalmology, vision science, microscopy, optical communications and beam control. This review provides a basic introduction to adaptive optics and a summary of the multidisciplinary articles included in this issue.

Early stage dental caries detection using near infrared spatial frequency domain imaging.

Early stage dental caries can be remineralized without the need for "drill-and-fill" treatments that are more invasive and less permanent. However, early stage caries lesions typically present as a white spot on a white background, resulting in many lesions only being identified after they have developed beyond the point of remineralization as cavities. We present a spatial frequency domain imaging technique to characterize the optical properties of dental tissue. This technique enables different dental tissue types (healthy enamel, healthy dentin and damaged or demineralized enamel) to be easily distinguished from one another and allows quantification of the reduced scattering coefficients of dental tissue. The use of near-infrared light at 850 nm allows high depth penetration into the tissue and suppression of absorption effects, ensuring only changes in the reduced scattering coefficient that result directly from demineralization of enamel are observed and simplifying the analysis method. This technique provides a tool to both guide the attention of dentists to areas of interest and potential demineralization, and to provide longitudinal quantified assessments to monitor caries lesion behaviour over time.

Three-dimensional data capture and analysis of intact eye lenses evidences emmetropia-associated changes in epithelial cell organization.

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4-6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations.

Cellular localisation of structurally diverse diphenylacetylene fluorophores.

Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor-acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed, compounds with high log D values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly lipophilic compounds (log D = 2-3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.

Multi-plane remote refocusing epifluorescence microscopy to image dynamic Ca 2 + events.

Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocusing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15 µ m, with a mean lateral stability of ≤ 0.32 µ m displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high framerate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca 2 + activity of the endothelial cell layer independently of the smooth muscle layer for several minutes.

Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene.

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Scattering of spoof surface plasmon polaritons in defect-rich THz waveguides.

We report on the first observation of 'Spoof' Surface Plasmon Polariton (SPP) scattering from surface defects on metal-coated 3D printed, corrugated THz waveguiding surfaces. Surface defects, a result of the printing process, are shown to assist the direct coupling of the incident free-space radiation into a spoof SPP wave; removing the need to bridge the photon momentum gap using knife-edge or prism coupling. The free space characteristics, propagation losses and confinement of the spoof SPPs to the surface are measured, and the results are compared to finite-difference time domain simulations. Angular resolved THz spectroscopy measurements reveal the scattering patterns from surfaces and are compared with Mie theory, taking into account the shortened wavelength of the photons in their bound SPP state compared to their free space wavelength. These results confirm yet another similarity between the properties of THz spoof SPPs and their natural, non-spoof, counterparts at optical and infrared frequencies which also, unexpectedly, adds functionality to the structures.

VasoTracker, a Low-Cost and Open Source Pressure Myograph System for Vascular Physiology.

Pressure myography, one of the most commonly used techniques in vascular research, measures the diameter of isolated, pressurized arteries to assess the functional activity of smooth muscle and endothelial cells. Despite the widespread adoption of this technique for assessing vascular function, there are only a small number of commercial systems and these are expensive. Here, we introduce a complete, open source pressure myograph system and analysis software, VasoTracker, that can be set-up for approximately 10% of the cost of commercial alternatives. We report on the development of VasoTracker and demonstrate its ability to assess various components of vascular reactivity. A unique feature of the VasoTracker platform is the publicly accessible website (http://www.vasotracker.com/) that documents how to assemble and use this affordable, adaptable, and expandable pressure myograph.

High-speed dual color fluorescence lifetime endomicroscopy for highly-multiplexed pulmonary diagnostic applications and detection of labeled bacteria.

We present a dual-color laser scanning endomicroscope capable of fluorescence lifetime endomicroscopy at one frame per second (FPS). The scanning system uses a coherent imaging fiber with 30,000 cores. High-speed lifetime imaging is achieved by distributing the signal over an array of 1024 parallel single-photon avalanche diode detectors (SPADs), minimizing detection dead-time maximizing the number of photons detected per excitation pulse without photon pile-up to achieve the high frame rate. This also enables dual color fluorescence imaging by temporally shifting the dual excitation lasers, with respect to each other, to separate the two spectrally distinct fluorescent decays in time. Combining the temporal encoding, to provide spectral separation, with lifetime measurements we show a one FPS, multi-channel endomicroscopy platform for clinical applications and diagnosis. We demonstrate the potential of the system by imaging SmartProbe labeled bacteria in ex vivo samples of human lung using lifetime to differentiate bacterial fluorescence from the strong background lung autofluorescence which was used to provide structural information.

Mitochondrial ATP production provides long-range control of endothelial inositol trisphosphate-evoked calcium signaling.

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.

Spatially structured cell populations process multiple sensory signals in parallel in intact vascular endothelium.

Blood flow, blood clotting, angiogenesis, vascular permeability, and vascular remodeling are each controlled by a large number of variable, noisy, and interacting chemical inputs to the vascular endothelium. The endothelium processes the entirety of the chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations that determine physiological output. Processing this enormous quantity of information is a major challenge facing the endothelium. We analyzed the responses of hundreds of endothelial cells to carbachol (CCh) and adenosine triphosphate (ATP) and found that the endothelium segregates the responses to these two distinct components of the chemical environment into separate streams of complementary information that are processed in parallel. Sensitivities to CCh and ATP mapped to different clusters of cells, and each agonist generated distinct signal patterns. The distinct signals were features of agonist activation rather than properties of the cells themselves. When there was more than one stimulus present, the cells communicated and combined inputs to generate new distinct signals that were nonlinear combinations of the inputs. Our results demonstrate that the endothelium is a structured, collaborative sensory network that simplifies the complex environment using separate cell clusters that are sensitive to distinct aspects of the overall biochemical environment and interactively compute signals from diverse but interrelated chemical inputs. These features enable the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable.

Tandem fluorescence and Raman (fluoRaman) characterisation of a novel photosensitiser in colorectal cancer cell line SW480.

The development of new imaging tools, molecules and modalities is crucial to understanding biological processes and the localised cellular impact of bioactive compounds. A small molecule photosensitiser, DC473, has been designed to be both highly fluorescent and to exhibit a strong Raman signal in the cell-silent region of the Raman spectrum due to a diphenylacetylene structure. DC473 has been utilised to perform a range of novel tandem fluorescence and Raman (fluoRaman) imaging experiments, enabling a thorough examination of the compound's cellular localisation, exemplified in colorectal cancer cells (SW480). This multifunctional fluoRaman imaging modality revealed the presence of the compound in lipid droplets and only a weak signal in the cytosol, by both Raman and fluorescence imaging. In addition, Raman microscopy detected the compound in a cell compartment we labelled as the nucleolus, whereas fluorescence microscopy did not detect the fluoRaman probe due to solvatochromatic effects in a local polar environment. This last finding was only possible with the use of tandem confocal Raman and fluorescence methods. By following the approach detailed herein, incorporation of strong Raman functional groups into fluorophores can enable a plethora of fluoRaman experiments, shedding further light on potential drug compound's cellular behaviour and biological activity.

Non-invasive in vivo quantification of the developing optical properties and graded index of the embryonic eye lens using SPIM.

Graded refractive index lenses are inherent to advanced visual systems in animals. By understanding their formation and local optical properties, significant potential for improved ocular healthcare may be realized. We report a novel technique measuring the developing optical power of the eye lens, in a living animal, by exploiting the orthogonal imaging modality of a selective plane illumination microscope (SPIM). We have quantified the maturation of the lenticular refractive index at three different visible wavelengths using a combined imaging and ray tracing approach. We demonstrate that the method can be used with transgenic and vital dye labeling as well as with both fixed and living animals. Using a key eye lens morphogen and its inhibitor, we have measured their effects both on lens size and on refractive index. Our technique provides insights into the mechanisms involved in the development of this natural graded index micro-lens and its associated optical properties.