This PDF file contains the front matter associated with SPIE Proceedings Volume 10013, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.

Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

A formidable challenge in modern respiratory healthcare is the accurate and timely diagnosis of lung infection and inflammation. The EPSRC Interdisciplinary Research Collaboration (IRC) ‘Proteus’ seeks to address this challenge by developing an optical fibre based healthcare technology platform that combines physiological sensing with multiplexed optical molecular imaging. This technology will enable in situ measurements deep in the human lung allowing the assessment of tissue function and characterization of the unique signatures of pulmonary disease and is illustrated here with our in-man application of Optical Imaging SmartProbes and our first device Versicolour.

Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H₂O₂) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H₂O₂ using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H₂O₂ to non-invasively detect H₂O₂ near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H₂O₂ and pH that is functional at biologically concentrations of H₂O₂ and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.

In this work we report characterization of bacterial biofilm using gold sputtered optical fiber probe as substrates for confocal Raman spectroscopy measurements. The chemical composition and the heterogeneity of biofilms in the extracellular polymeric substances (EPS) was evaluated. The spatial distribution of bacterial biofilm on the substrates during their growth phase was studied using Raman imaging. Further, the influence of substrate’s surface on bacterial adhesion was investigated by studying growth of biofilms on surfaces with hydrophilic and hydrophobic coatings. This study validates the use of gold sputtered optical fiber probes as SERS substrates in confocal microscopic configuration to identify and characterize clinically relevant biofilms.

Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

Vitamin B12 (cobalamin and its derivatives) deficiency has been identified as a potential modifiable risk factor for dementia and Alzheimer’s disease. Chronic deficiency of vitamin B12 has been significantly associated with an increased risk of cognitive decline. An effective and efficient method for measuring vitamin B12 concentration in human blood would enable ongoing tracking and assessment of this potential modifiable risk factor. In this work we present an optical sensor based on resonance Raman spectroscopy for rapid measurements of vitamin B12 in human blood serum. The measurement takes less than a minute and requires minimum preparation (centrifuging) of the collected blood samples.

We report on the development of an optical fiber based probe for in vivo measurements of brain temperature. By using a thin layer of rare-earth doped tellurite glass on the tip of a silica optical fiber a durable probe, suitable for long-term in vivo measurements of temperature can be fabricated. The probe can be interrogated using a portable optical measurement setup, allowing for measurements to be performed outside of standard optical laboratories as no alignment of components is required. This setup is deployed to a medical research laboratory to show preliminary results on the use of these optical fibers for in vivo pre-clinical measurements of brain temperature.

Multielectrode arrays are a powerful tool for recording biopotentials, however they are limited by issues related to wiring complexity and channel-count. We present a novel concept for a liquid crystal-based optical electrode (optrode) that does not require the electrical circuitry associated with reading and amplifying each channel, thus providing superior spatial resolution and signal-to-noise ratio. Through computational modeling, we show that it is possible to accurately image biopotentials by coupling them to the electrodes of a LC cell and measuring their re ectance under parallel polarisers.

An evanescent wave absorbance (EWA) based U-bent fiberoptic sandwich immunobiosensor with human IgG detection limits of 6.67 fM (1 pg/ml of human IgG) and 66.7 aM (10 fg/ml of HIgG) is demonstrated by exploiting immunogold labels and subsequently silver enhancement respectively. Such very low detection limits were achieved with the help of enhanced evanescent filed at the bend region of U-bent optical fiber probe that allows efficient interaction of light with 40 nm immunogold labels on the probe surface resulting in measurable optical absorbance changes. The other significant advantages of the demonstrated sensing scheme are low cost optoelectronic instrumentation consisting of an commercial green LED and a photodetector (S150C, Thorlabs Inc.), small volumes of sample and immunogold reagent each of 25 μl and rapid detection in 20 min. These results from the plasmonic fiberoptic biosensor demonstrate its huge potential for development of point-of-care diagnostic devices for sensitive and rapid detection of analytes.

Bio-imaging requires robust ultra-bright probes without causing any toxicity to the cellular environment, maintain their stability and are chemically inert. In this work we present hexagonal boron nitride (hBN) nanoflakes which exhibit narrowband ultra-bright single photon emitters1. The emitters are optically stable at room temperature and under ambient environment. hBN has also been noted to be noncytotoxic and seen significant advances in functionalization with biomolecules2,3. We further demonstrate two methods of engineering this new range of extremely robust multicolour emitters across the visible and near infrared spectral ranges for large scale sensing and biolabeling applications.

The rapid development of a variety of molecular contrast agents makes the multimodality bioimaging highly attractive towards higher resolution, more sensitive, informative diagnosis. The key lies in the development of facile material synthesis that allows the integration of multiple contrast agents, ideally in a way that each of the components should be logically assembled to maximize their performances. Here, we report the one-pot programmable growth of multifunctional heterogeneous nanocrystal with tunable size, shape, composition, and properties. We demonstrated a facile one-pot hot-injection method to enable the highly selectively controlled growth of different sodium lanthanide fluoride nanomaterials in either longitudinal or transversal directions with atomic scale precision. This technique allows the upconversion luminescence signal, MRI signal and x-ray signal logically integrated and optimized within one single versatile nanoplatform for multimode bioimaging. These findings suggest that the facile strategy developed here have the promising to get the desired heterogeneous nanocrystals as an all-in-one contrast agent for integrated and self-correlative multimodal bioimaging.

DNA is a biocompatible scaffold that allows for the design of a variety of nanostructures, from straightforward double stranded DNA to more complex DNA origami and 3-D structures. By modifying the structures, with dyes, nanoparticles, or enzymes, they can be used to create light harvesting and energy transfer systems. We have focused on using Förster resonance energy transfer (FRET) between organic fluorophores separated with nanometer precision based on the DNAs defined positioning. Using FRET theory we can control the direction of the energy flow and optimize the design parameters to increase the systems efficiency. The design parameters include fluorophore selection, separation, number, and orientation among others. Additionally the use of bioluminescence resonance energy transfer (BRET) allowed the use of chemical energy, as opposed to photonic, to activate the systems. Here we discuss a variety of systems, such as the longest reported DNA-based molecular photonic wires (> 30 nm), dendrimeric light harvesting systems, and semiconductor nanocrystals integrated systems where they act as both scaffold and antennae for the original excitation. Using a variety of techniques, a comparison of different types of structures as well as heterogeneous vs. homogenous FRET was realized.

For decades Surface Plasmon Resonance (SPR) has been one of the corner stones of label free biosensing with a wide range of architectures including optical fiber based SPR. Traditionally, the resonance is monitored through reflectivity measurements at a single wavelength as a function of the incident angle in a standard Kretschmann configuration, or transmission of broadband light through an optical fiber. In both cases, SPR is inferred through optical losses. An alternative approach is to use SPR scattering induced by rough metallic coatings, enabling to turn an intrinsically nonradiative process into a radiative one. As a result, the SPR signal corresponding to the resonance can be seen as light at specific wavelengths being re-emitted by the rough metallic coating.

Here, we present results we have achieved using SPR scattering as an alternative approach for optical fiber based plasmonic sensors. Although the use of a rough metallic coating induces some inherent limitations, such as a lower resolution, the architectural advantages and simplicity of the approach offer additional opportunities, such as multiplexing and self-referencing, which are not possible otherwise with a single fiber SPR sensor. A way to overcome the lower resolution that involves the use of microstructured optical fibers, as well as a new perspective on a complementary application, such as Metal Enhanced Fluorescence, which greatly benefits from SPR scattering, will be presented.

The small dimensions of optical fiber sensors are of interest to biological applications, given the ability to penetrate relatively inaccessible regions. However, conventional optical fibers are larger than biological material such as cells, and thus there is a need for further miniaturization. Here we present the fabrication of ultra-small Fabry-Perot cavities written into optical micro-fibers using focused ion beam (FIB) milling. We have fabricated cavities as small as 2.8 μm and demonstrated their use for measuring refractive index. In order to achieve sensitive measurements we interrogate at visible wavelengths, thereby reducing the free spectral range of the interferometer (relative to infra-red interrogation), increasing the number of interference fringes, and allowing for the implementation of the Fourier shift method.

Elementary PDMS-based building blocks of fluidic, optical and optofluidic components for Lab on a chip (LOC) platforms has here been developed. All individual modules are compatible and can be anchored and released with the help of puzzle-type connectors This approach is a powerful toolbox to create modular optofluidic systems (MOPS), which can be modified/upgraded to user needs and in-situ reconfigurable. In addition, the PDMS can locally be functionalized, defining a modular biosensor. Measurements in absorbance and fluorescence have been pursued as demonstrator.

Optical fiber sensors for Raman spectroscopy based on hollow core optical fibers have shown great promise due to their low glass background, and high signal collection efficiency. We have previously demonstrated how glass billet extrusion can be used to make simplified hollow core fibers based on a single suspended ring. In this work we investigate the performance of these optical fibers as sensors for Raman spectroscopy. These fibers are used to excite samples at a range of laser excitation wavelengths to scan across the transmission profile of the optical fibers, allowing comparison of the performance of these fibers against commercially-available alternatives.

Whispering gallery modes (WGMs) allow for remarkable refractive index sensing performance with extremely low detection limits, and thus have found use in various emerging label free biosensing applications. Among the different types of resonators which have been studied, microcapillaries have the unique property of having the evanescent fields extend into and sample the medium inside the resonator, which is particularly interesting because the resonator itself serves as a microfluidic channel.

Here, lasing of the WGMs in fluorescent microcapillaries is demonstrated for the first time, and their application to refractive index sensing is investigated. The laser gain medium used here is embedded inside a high refractive index polymer coating deposited onto the inner surface of the capillary. Lasing can only be realized for thick polymer coatings (in this case ≥ 800 nm), with higher Q factor but also stronger confinement of the propagating wave, which lowers the refractive index sensitivity compared to non-lasing capillaries which can have thinner polymer coatings. We however find that the large improvement in signal-to-noise ratio and Q factor realized upon lasing more than compensates for the reduced sensitivity, resulting in an order-of-magnitude improvement in the detection limit for refractive index sensing.

Estimation of renal function and drug renal disposition in acute kidney injury (AKI), is important for appropriate dosing of drugs and adjustment of therapeutic strategies, but is challenging due to fluctuations in kidney function. Multiphoton microscopy has been shown to be a useful tool in studying drug disposition in liver and can reflect dynamic changes of liver function. We extend this imaging technique to investigate glomerular filtration rate (GFR) and tubular transporter functional change in various animal models of AKI, which mimic a broad range of causes of AKI such as hypoxia (renal ischemia- reperfusion), therapeutic drugs (e.g. cisplatin), rhabdomyolysis (e.g. glycerol-induced) and sepsis (e.g. LPSinduced). The MPM images revealed acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation in AKI groups compared to control group. In control animal, systemically injected FITC-labelled inulin was rapidly cleared from glomerulus, while the clearance of FITC-inulin was significantly delayed in most of animals in AKI group, which may reflect the reduced GFR in AKI. Following intravenous injection, rhodamine 123, a fluorescent substrate of p-glycoprotein (one of tubular transporter), was excreted into urine in proximal tubule via p-glycoprotein; in response to AKI, rhodamine 123 was retained in tubular cells as revealed by slower decay of fluorescence intensity, indicating P-gp transporter dysfunction in AKI. Thus, real-time changes in GFR and transporter function can be imaged in rodent kidney with AKI using multiphoton excitation of exogenously injected fluorescent markers.

Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid “off-on” fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

Paracetamol is the most readily available and widely used painkiller. However, its toxicity remains the most common cause of liver injury. The toxicity of paracetamol has been attributing to its toxic metabolite, which depletes cellular glutathione (GSH) stores and reacts within cells to increase oxidative stress, leading to mitochondrial dysfunction and cell necrosis. Multiphoton microscopy (MPM) and fluorescence lifetime imaging (FLIM) can provide quantitative imaging of biological tissues and organs in vivo and allow direct visualization of cellular events, which were used to monitor cellular metabolism in paracetamol-induced toxicity in this study. To better understand mechanisms of paracetamol induced liver injury, the redox ratio of NADH/FAD in liver cells were detected and quantified by MPM imaging to represent the relative rates of glycolysis and oxidative phosphorylation within cells. Compared to normal liver, average fluorescence lifetime of NADH and redox ratio of NADH/FAD in hepatocytes was significantly decreased after paracetamol overdose for 12 and 24 hrs, reflecting impaired metabolic activity. GSH levels of treatment groups were significantly lower than those of normal livers, with gradually decreasing from periportal to centrilobular zonation. This imaging technique has significant implications for investigating metabolic mechanisms of paracetamol toxicity.

Chronic liver disease causes 2,000 deaths in Australia per year and early diagnosis is crucial to avoid progression to cirrhosis and end stage liver disease. There is no ideal method to evaluate liver function. Blood tests and liver biopsies provide spot examinations and are unable to track changes in function quickly. Therefore better techniques are needed. Non-invasive imaging has the potential to extract increased information over a large sampling area, continuously tracking dynamic changes in liver function. This project aimed to study the ability of three imaging techniques, multiphoton and fluorescence lifetime imaging microscopy, infrared thermography and photoacoustic imaging, in measuring liver function. Collagen deposition was obvious in multiphoton and fluorescence lifetime imaging in fibrosis and cirrhosis and comparable to conventional histology. Infrared thermography revealed a significantly increased liver temperature in hepatocellular carcinoma. In addition, multiphoton and fluorescence lifetime imaging and photoacoustic imaging could both track uptake and excretion of indocyanine green in rat liver. These results prove that non-invasive imaging can extract crucial information about the liver continuously over time and has the potential to be translated into clinic in the assessment of liver disease.

In this study, poly(lactic-co-glycolic acid) (PLGA) nanocomposites were developed by incorporating a photosensitizer, verteporfin and gold nanoparticles into this polymeric matrix and utilised for enhanced photoynamic therapy. Both enhanced fluorescence and singlet oxygen generation from verteporfin were observed in this new formulation under both 425nm LED and 405nm laser illumination. A maximum enhancement factor of 2.5 for fluorescence and 1.84 for ¹O₂ generation was obtained when the molar ratio of gold:VP was 5:1 and excited at 425 nm, compared with PLGA doped with verteporfin only. The experiment results could be explained by the local electric field enhancement of gold nanoparticles. Furthermore, in vitro cell-killing effect on human pancreatic cancer cells was also demonstrated by using this new formulation following light exposure, indicating the utility of these nanocomposites for enhanced photodynamic therapy.

Biomonitoring studies apply biological responses of sensitive biomonitor organisms to rapidly detect adverse environmental changes such as presence of physic-chemical stressors and toxins. Behavioral responses such as changes in swimming patterns of small aquatic invertebrates are emerging as sensitive endpoints to monitor aquatic pollution. Although behavioral responses do not deliver information on an exact type or the intensity of toxicants present in water samples, they could provide orders of magnitude higher sensitivity than lethal endpoints such as mortality. Despite the advantages of behavioral biotests performed on sentinel organisms, their wider application in real-time and near realtime biomonitoring of water quality is limited by the lack of dedicated and automated video-microscopy systems. Current behavioral analysis systems rely mostly on static test conditions and manual procedures that are time-consuming and labor intensive. Tracking and precise quantification of locomotory activities of multiple small aquatic organisms requires high-resolution optical data recording. This is often problematic due to small size of fast moving animals and limitations of culture vessels that are not specially designed for video data recording. In this work, we capitalized on recent advances in miniaturized CMOS cameras, high resolution optics and biomicrofluidic technologies to develop near real-time water quality sensing using locomotory activities of small marine invertebrates. We present proof-of-concept integration of high-resolution time-resolved video recording system and high-throughput miniaturized perfusion biomicrofluidic platform for optical tracking of nauplii of marine crustacean Artemia franciscana. Preliminary data demonstrate that Artemia sp. exhibits rapid alterations of swimming patterns in response to toxicant exposure. The combination of video-microscopy and biomicrofluidic platform facilitated straightforward recording of fast moving objects. We envisage that prospectively such system can be scaled up to perform high-throughput water quality sensing in a robotic biomonitoring facility.

The enzyme horseradish peroxidase (HRP) has been extensively used in biochemistry for its ability to amplify a weak signal. By using HRP catalyzed substrate as the gain medium, we demonstrate sensitive ion concentration detection based on the optofluidic laser. The enzyme catalyzed reaction occurs in bulk solution inside a Fabry-Perot laser cavity, where the colorless, non-fluorescent 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) substrate is oxidized to produce highly fluorescent resorufin. Laser emission is achieved when pumped with the second harmonic wave of a Q-switched YAG laser. Further, we use sulfide anion (S^2-) as an example to investigate the sensing performance of enzyme catalyzed optofluidic laser. The laser onset time difference between the sample to be tested and the reference is set to be the sensing output. Thanks to the amplification effects of both the enzymatic reaction and laser emission, we achieve a detection limit of 10 nM and a dynamic range of 3 orders of magnitude.

A fluorometric acetone biosniffer (biochemical gas sensor) for assessment of lipid metabolism utilizing reverse reaction of secondary alcohol dehydrogenase was constructed and evaluated. The biosniffer showed highly sensitivity and selectivity for continuous monitoring of gaseous acetone. The measurement of breath acetone concentration during fasting and aerobic exercise were also investigated. The acetone biosniffer provides a novel analytical tool for noninvasive evaluation of human lipid metabolism and it is also expected to use for the clinical and physiological applications such as monitoring the progression of diabetes.

This work aims to develop a stable, compact and portable fibre optic sensing system which is capable of real time detection of the mercury ion (II), Hg²⁺. A novel fluorescent polymeric material for Hg²⁺ detection, based on a coumarin derivative (acting as the fluorophore) and an azathia crown ether moiety (acting as the mercury ion receptor), has been designed and synthesized. The material was covalently attached to the distal end of an optical fibre and exhibited a significant increase in fluorescence intensity in response to Hg²⁺ in the μM concentration range via a photoinduced electron transfer (PET) mechanism. The sensor has also demonstrated a high selectivity for Hg²⁺ over other metal ions. A washing protocol was identified for sensor regeneration, allowing the probe to be re-used. The approach developed in this work can also be used for the preparation of sensors for other heavy metals.

We designed and fabricated a smart and stimuli responsive membrane to cater on demand molecular transporting applications. A novel photoswitchable peptide (PSP) was synthesized and attached inside nanoporous anodic alumina membranes (NAAMs) pores. The PSP specifically switched between its cis and trans photostationary states on exposure to 364 nm and 440 nm wavelength lights respectively, which not only provided the ability to control its pore diameter but also the surface chemistry. The switchable molecular transport properties of the PSP-NAAMs have been shown as a function of the light exposure. Most importantly, the molecular transport across PSP-NAAMs could be repeatedly switched between on and off state, which is highly significant for on-demand triggered drug release systems.

Spiropyran-based fluorescence sensors are an ideal target for intracellular metal ion sensing, due to their biocompatibility, red emission frequency and photo-controlled reversible analyte binding for continuous signal monitoring. However, increasing the brightness of spiropyran-based sensors would extend their sensing capability for live-cell imaging. In this work we look to enhance the fluorescence of spiropyran-based sensors, by incorporating an additional fluorophore into the sensor design. We report a 5-membered monoazacrown bearing spiropyran with metal ion specificity, modified to incorporate the pyrene fluorophore. The effect of N-indole pyrene modification on the behavior of the spiropyran molecule is explored, with absorbance and fluorescence emission characterization. This first generation sensor provides an insight into fluorescence-enhancement of spiropyran molecules.

Near-infrared spectroscopy (NIRS) has been used in medical imaging to obtain oxygenation and hemodynamic response in the cerebral cortex. This technique has been applied in cortical activation detection and functional connectivity in brain research. Despite some advances in functional connectivity, most of the studies have focused on the prefrontal cortex and little has been done to study the somatosensory region (S1). For that reason, the aim of our present study is to assess bilateral connectivity in the somatosensory region by using NIRS and noxious stimulation. Eleven healthy subjects were investigated using near-infrared spectroscopy during an acupuncture stimulation procedure to safely induce pain in subjects. A multiscale analysis based on wavelet transform coherence (WTC) was designed to assess the functional connectivity of corresponding channel pairs within the left and right s1 region. The cortical activation in the somatosensory region was higher after the acupuncture stimulation, which was consistent with similar studies. The coherence in time-frequency domain between homologous signals generated by contralateral channel pairs revealed two main periods (3.2 s and 12.8 s) with high coherence. Based on the WTC analysis, it was also found that the coherence increase in these periods was task-related. This study contributes to the research field to investigate cerebral hemodynamic response of pain perception using NIRS and demonstrates the use of wavelet transform as a method to investigate functional lateralization in the cerebral cortex.

Wearable computing devices, e.g. Google Glass, Smart watch, embodies the new human design frontier, where technology interfaces seamlessly with human gestures. During examination of any subject in the field (clinic, surgery, agriculture, field survey, water collection), our sensory peripherals (touch and vision) often go hand-in-hand. The sensitivity and maneuverability of the human fingers are guided with tight distribution of biological nerve cells, which perform fine motor manipulation over a range of complex surfaces that is often out of sight. Our sight (or naked vision), on the other hand, is generally restricted to line of sight that is ill-suited to view around corner. Hence, conventional imaging methods are often resort to complex light guide designs (periscope, endoscopes etc) to navigate over obstructed surfaces. Using modular design strategies, we constructed a prototype miniature microscope system that is incorporated onto a wearable fixture (thimble). This unique platform allows users to maneuver around a sample and take high resolution microscopic images. In this paper, we provide an exposition of methods to achieve a thimble microscopy; microscope lens fabrication, thimble design, integration of miniature camera and liquid crystal display.

Despite the growing demand and numerous applications for the biomedical community, the developments in millifluidic devices for small model organisms are limited compared to other fields of biomicrofluidics. The main reasons for this stagnanation are difficulties in prototyping of millimeter scale and high aspect ratio devices needed for large metazoan organisms. Standard photolithography is in this context a time consuming procedure not easily adapted for fabrication of molds with vertical dimensions above 1 mm. Moreover, photolithography is still largely unattainable to a gross majority of biomedical laboratories willing to pursue custom development of their own chip-based platforms due to costs and need for dedicated clean room facilities. In this work, we present application of high-definition additive manufacturing systems for fabrication of 3D printed moulds used in soft lithography. Combination of 3D printing with PDMS replica molding appears to be an alternative for millifluidic systems that yields rapid and cost effective prototyping pipeline. We investigated the important aspects on both 3D printed moulds and PDMS replicas such as geometric accuracies and surface topology. Our results demonstrated that SLA technologies could be applied for rapid and accurate fabrication of millifluidic devices for trapping of millimetre-sized specimens such as living zebrafish larvae. We applied the new manufacturing method in a proof-of-concept prototype device capable of trapping and immobilizing living zebrafish larvae for recording heart rate variation in cardio-toxicity experiments.

The fish embryo toxicity (FET) biotest performed on embryos of zebrafish (Danio rerio) has gained significant popularity as a rapid and inexpensive alternative approach in chemical hazard and risk assessment. The FET was designed to evaluate acute toxicity on embryonic stages of fish exposed to the test chemical. The current standard, similar to most traditional methods for evaluating aquatic toxicity provides, however, little understanding of effects of environmentally relevant concentrations of chemical stressors.

We postulate that significant environmental effects such as altered motor functions, physiological alterations reflected in heart rate, effects on development and reproduction can occur at sub-lethal concentrations well below than LC₁₀. Behavioral studies can, therefore, provide a valuable integrative link between physiological and ecological effects. Despite the advantages of behavioral analysis development of behavioral toxicity, biotests is greatly hampered by the lack of dedicated laboratory automation, in particular, user-friendly and automated video microscopy systems.

In this work we present a proof-of-concept development of an optical system capable of tracking embryonic vertebrates behavioral responses using automated and vastly miniaturized time-resolved video-microscopy. We have employed miniaturized CMOS cameras to perform high definition video recording and analysis of earliest vertebrate behavioral responses. The main objective was to develop a biocompatible embryo positioning structures that were suitable for high-throughput imaging as well as video capture and video analysis algorithms. This system should support the development of sub-lethal and behavioral markers for accelerated environmental monitoring.

Here we propose a region-recognition approach with iterative thresholding, which is adaptively tailored to extract the appropriate region or shape of spatial frequency. In order to justify the method, we tested it with different samples and imaging conditions (different objectives). We demonstrate that our method provides a useful method for rapid imaging of cellular dynamics in microfluidic and cell cultures.

Behavioural alterations can occur as a result of a toxicant exposure at concentrations significantly lower than lethal effects that are commonly measured in acute toxicity testing. The use of alternating light and dark photoperiods to test phototactic responses of aquatic invertebrates in the presence of environmental contaminants provides an attractive analytical avenue. Quantification of phototactic responses represents a sublethal endpoint that can be employed as an early warning signal. Despite the benefits associated with the assessment of these endpoints, there is currently a lack of automated and miniaturized bioanalytical technologies to implement the development of toxicity testing with small aquatic species. In this study we present a proof-of-concept microfluidic Lab-on-a-Chip (LOC) platform for the assessment of rotifer swimming behavior in the presence of the toxicant copper sulfate. The device was designed to assess impact of toxicants at sub-lethal concentrations on freshwater crustacean Brachionus calyciflorus, testing behavioral endpoints such as animal swimming distance, speed and acceleration. The LOC device presented in this work enabled straightforward caging of microscopic crustaceans as well as non-invasive analysis of rapidly swimming animals in a focal plane of a video-microscopy system. The chip-based technology was fabricated using a new photolithography method that enabled formation of thick photoresist layers with minimal distortion. Photoresist molds were then employed for replica molding of LOC devices with poly(dimethylsiloxane) (PDMS) elastomer. The complete bioanalytical system consisted of: (i) microfluidic PDMS chip-based device; (ii) peristaltic microperfusion pumping manifold; (iii) miniaturized CMOS camera for video data acquisition; and (iv) video analysis software algorithms for quantification of changes in swimming behaviour of B. calyciflorus in response to reference toxicants.

We present recent results on the realisation and demonstration of an integrated optofluidic lab-on-a-chip measurement system. The system consists of an integrated on-chip automated microfluidic fluid handling subsystem, coupled with bimodal nano-interferometer waveguide technology, and is applied in the context of detection of antibiotics in seawater. The bimodal waveguide (BMWG) is a highly sensitive label-free biosensor. Integration of complex microfluidic systems with bimodal waveguide technology enables on-chip sample handling and fluid processing capabilities and allows for significant automation of experimental processes. The on-chip fluid-handling subsystem is realised through the integration of pneumatically actuated elastomer pumps and valves, enabling high temporal resolution sample and reagent delivery and facilitating multiplexed detection processes.

Nanofabrication of metamaterials based on thin metallic films have resulted in a host of different designs that support Extraordinary Optical Transmission (EOT). In contrast to the more widely studied circular-shaped apertures, cross-shaped apertures have characteristics which can be modified in response to linearly polarized light, opening up new pathways for light manipulation at the nanoscale. Here we present a systematic study of the influence of device geometry and composition on the functional characteristics of polarisation controlled optical plasmonic devices. We also discuss some issues that arise using the focused ion beam (FIB) milling technique to fabricate optical metamaterials. In particular, we show that producing high-quality patterns lead to a significant over-deposition of the substrate material. This effect significantly alters the metal surface chemistry, which poses a considerable obstacle for applications involving molecular and bio sensing. This work lays the foundation for the optimisation of the properties of optical plasmonic devices for a wide-range of applications including colour filtering and bio-sensing.

Background: Upconverting nanoparticles (UCNPs) represent a unique class of nanomaterials, able to convert infrared excitation light into long lifetime visible and infrared photoluminescence, within the “optical transparency window” of biological tissues. This makes UCNPs an attractive contrast agent for background-free bioimaging. However, assynthesized UCNPs are hydrophobic and need additional surface coating for stability in water-based solutions and further functionalization. Polyethylenimine (PEI), a polycationic amphiphilic polymer, is a well-known transfection agent for gene delivery and a popular material for UCNPs surface hydrophilization. Combining the functional properties of UCNPs and PEI is extremely useful for precise visualization of genetic manipulations and intracellular drug delivery. At the same time, PEI is toxic to cells, while the photoluminescent properties of UCNPs are very sensitive to surface chemistry and environment. Then, creation of hydrophilic, biocompatible and simultaneously bright UCNPs, modified by PEI (UCNP-PEI), is a challenging task.

Objectives: To analyze the effects of multilayer shielding coatings on cytotoxicity, cellular uptake and photoluminescent properties of UCNP-PEI.

Methods and results: UCNP-PEI were modified with additional two or three layers of various polymers and characterized by size, surface charge and photophysical properties. HaCaT keratinocytes were exposed to the particles for 24 or 120 h to study the cytotoxicity and cellular uptake. The results show that onion-like coatings of UCNP-PEI simultaneously decrease cytotoxicity and relative luminescence of the particles, depending on structure and method of formation of multilayer coating.

Conclusions: Rational design of UCNP-PEI using extra coatings layers can help to keep acceptable levels of biocompatibility and photoluminescence intensity.

The main objective of our work was to design a light source which should be capable to collect and illuminate light of LEDs at the smaller aperture of cone (9mm) which could be either coupled with secondary optics of a microscope or utilized independently for hyperspectral studies. Optimized performance of cone was assessed for different substrates (diffused glass silica, Alumina, Zerodur glass, acrylic plastic) and coating surfaces (white diffused, flat white paint, standard mirror) using a simulation software. The parameters optimized for truncated cone include slanting length and Top Major R (Larger diameter of cone) which were also varied from 10 to 350 mm and 10 to 80 mm respectively. In order to see affect of LED positions on cone efficiency, the positions of LED were varied from central axis to off-axis. Similarly, interLED distance was varied from 2 mm to 6 mm to reckon its effect on the performance of cone. The optimized Slant length (80 mm) and Top Major R (50 mm) were determined for substrates (glass zerodur or acrylic plastic) and coating surface (standard mirror). The output profile of truncated source was found non uniform, which is a typical presentation of non imaging optics problem. The maximum efficiency of cone has been found for LED at the centre and it was found decreasing as LED moves away from the central axis. Moreover, shorter the interLED distance, better is the performance of cone. The primary optics of cone shaped light source is capable to lit visible and UV LEDs in practical design. The optimum parameters obtained through simulations could be implemented in the fabrication procedure if the reflectance of source would have been maintained upto finish level of a standard mirror.

Traditional spectral imaging systems mainly rely on spatial scanning or spectral scanning methods to acquire spatial and spectral features. The acquisition is time-consuming and cannot fully satisfy the need of monitoring dynamic phenomenon and observing different structures of the specimen simultaneously. To overcome these barriers, we develop a video-rate simultaneous multispectral imaging system built with a spectral multiplexed volume holographic grating (VHG) and few optical components. Four spectral multiplexed volume holograms optimized for four discrete spectral bands (centered at 488 nm, 530 nm, 590 nm and 620 nm) are recorded into an 8×12 mm photo-thermal refractive glass. The diffraction efficiencies of all the holograms within the multiplexed VHG are greater than 80%. With the high throughout multiplexed VHG, the system can work with both reflection and fluorescence modes and allow simultaneous acquisition of spectral and spatial information with a single exposure. Imaging experiments demonstrate that the multispectral images of the target illuminated with white light source can be obtained. Fluorescence images of multiple fluorescence objects (two glass beads filled with 20 uL 1.0 mg/mL quantum dots solutions that emit 530 ± 15 nm and 620 ± 15 nm fluorescence, respectively) buried 3 mm below the surface of a tissue mimicking phantom are acquired. The results demonstrate that the system can provide complementary information in fluorescence imaging. The design diagram of the proposed system is given to explain the advantage of compactness and flexibility in integrating with other imaging platforms.

The photoisomerisation of azobenzenes between trans and cis results in well-defined changes in geometry and a considerable change of polarity. Thus, incorporating an azobenezene into a bioactive compound provides an opportunity to control biological activity, with ideally one configuration being active and the other inactive. This can allow the role of a specific biomolecule to be probed in its native environment by controlling activity both spatially and temporally using light. Incorporating such a photoswitchable moiety into the structure of a known GRK2 inhibitor can generate photoswitchable inhibitors, which can be used to reversibly regulate the activity of GRK2, and hence GPRCs.

Although whispering gallery mode (WGM) biosensors have shown tremendous potential, they are still yet to find practical use as biomedical diagnostic tools. This is primarily due to the nature of the interrogation mechanism itself which relies on indirect measurement of the binding of a specific biomolecule onto the sensor through the associated refractive index change. Since nonspecific binding cannot be differentiated from the specific interaction of interest, this can result in a high rate of false positive readings when the detection is performed in complex biological samples.

Here we show that this inherent limitation can be solved using a relatively simple approach. This approach involves the development of a self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a suspended core optical fiber. Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted human serum samples. The fiber allows remote excitation and collection of the WGMs of the microspheres in a dip sensing setting. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events, while the other microsphere is functionalized to detect the specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.

The crisis rate of brain tumor is about one point four in ten thousands. In general, cytotechnologists take charge of cytologic diagnosis. However, the number of cytotechnologists who can diagnose brain tumors is not sufficient, because of the necessity of highly specialized skill. Computer-Aided Diagnosis by computational image analysis may dissolve the shortage of experts and support objective pathological examinations. Our purpose is to support a diagnosis from a microscopy image of brain cortex and to identify brain tumor by medical image processing. In this study, we analyze Astrocytes that is a type of glia cell of central nerve system. It is not easy for an expert to discriminate brain tumor correctly since the difference between astrocytes and low grade astrocytoma (tumors formed from Astrocyte) is very slight. In this study, we present a novel method to segment cell regions robustly using BING objectness estimation and to classify brain tumors using deep convolutional neural networks (CNNs) constructed by deep residual learning. BING is a fast object detection method and we use pretrained BING model to detect brain cells. After that, we apply a sequence of post-processing like Voronoi diagram, binarization, watershed transform to obtain fine segmentation. For classification using CNNs, a usual way of data argumentation is applied to brain cells database. Experimental results showed 98.5% accuracy of classification and 98.2% accuracy of segmentation.

The present study intends to understand the importance of cellular marker in tissue regeneration regulated upon irradiation of low power laser light in burn inflicted mice. Under anesthetic conditions, the thermal injury was induced on Swiss albino mice of either sex. Following injury, the animals were randomly divided into three groups; i. e., un-illuminated control, the group treated with 5% Povidone iodine (reference standard) and single exposure of 3 J/cm² (830 nm). Burn tissue samples from each group were excised at day 6 post burn injury upon euthanization and used for histological and immunohistochemical analysis. Haematoxylin and Eosin (H and E) staining was performed on the selected sections to asses proliferation and angiogenesis at day 6 post-injury. For immunohistochemical analysis, tissue sections from all the three treatment groups on day 6 were stained using specific antibody against Proliferating cell nuclear antigen (PCNA). The results of the histological and immunohistochemical analysis showed improved tissue restoration in animals treated with optimal laser influence as compared to un-illuminated controls. The findings of present study clearly demonstrated the beneficial effects of 830 nm laser in burn wound healing and its influence in regulating the cellular marker.

Ptychography has recently been adapted for the recovery of the complete Jones matrix of an anisotropic specimen, using a vectorial form of the Ptychographic Iterative Engine (vPIE) for a set of linearly polarized probes. Here we show that this method can be applied to the recovery of the in-plane components of the elastic strain tensor in a diametrically compressed disc. The advantages and disadvantages of vPIE for the recovery of strain information from ‘real-world’ samples is discussed as well as the potential for this approach to be applied to the characterization of the mechanical properties of optically transparent materials

Digital holographic microscopy (DHM) is a powerful in-vitro biological imaging tool. In this paper, we report a fully automated off-axis digital holographic microscopy system completed with a graphical user interface in the Matlab © environment. The interface primarily includes Fourier domain processing, phase reconstruction, aberration compensation and autofocusing. A variety of imaging operations such as region of interest selection, de-noising mode (filtering and averaging), low frame rate imaging for immediate reconstruction and high frame rate imaging routine (~ 27 fps) are implemented to facilitate ease of use.

Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems.

We report on a new regime for optical trapping of diamond nanoparticles in liquid. While holding a nanodiamond (~150 nm) containing many NV centres (~10^3) at the focus of classical optical tweezers, we add a second laser slightly detuned from the dipole transition of the NVs. We measure a change in trap stiffness of ~10%. Remarkably, we show that our results must include collective effects – ‘superradiance’ – between colour centres, which has never been reported before for solid-state systems at room temperature. We discuss how these resonant forces depend on (and can be enhanced by) the type, number, dipole strength and spectral linewidth of the centres.

The traditional process of making glass lenses requires grinding and polishing of the material which is a tedious and sensitive process. Existing polymer lens making techniques, such as high temperature reflow techniques, have been significantly simple lens making processes which cater well to customer industry. Recently, the use of UV-curing liquid lens has ushered in customized lens making (Printed Optics), but contains undesirable yellowing effects. Polydimethylsiloxane (PDMS) is a transparent polymer curable at low temperature (<100°C) provides an alternative to lens making. In this work, we showed that PDMS lenses are fabricated using single silicone droplets which are formed in a guided and controlled passive manner using 3D printed tools. These silicone lenses have attributes such as smoothness of curvature, resilience to temperature change, low optical aberrations, high transparency (>95%) and minimal aging (yellowing). Moreover, these lenses have a range of focal lengths (3.5 mm to 14.5 mm as well as magnifications (up to 160X). In addition, we created smartphone attachment to turn smart device (tablet or smartphone) into a low-powered microscope. In future we plan to extend this method to produce microlens array.

Non-invasive optical screening mammography has a significant barrier in the extreme scatter of human tissue at optical wavelengths. A volume scanner suited for high numerical aperture capture of scattered light from diffuse media has been designed, modelled using Trace Pro software and experimentally constructed. Modelling results indicate the presence of an embedded volume with different scatter properties from the bulk yields a measurable difference in the overall scatter pattern and intensity recorded. Work towards a full tomographic reconstruction from scattered light recorded on the two dimensional array detector is currently underway.

Plasmonic devices provide a unique sensitivity to changes in the permittivity of the immediate, near-surface environment. In this work we explore the use of dual pitch plasmonic devices combined with microfluidics for polarization enhanced colour sensing of a chemicals’ refractive index. We demonstrate that the use of cross-shaped apertures can produce polarization tunable color based sensing in the optical regime and show that the spectral variations as a function of the incident polarization can be decomposed into contributions from the two orthogonal modes that characterize the dual pitch plasmonic device. Finally we demonstrate that the use of the full colour spectrum in the visible range in combination with polarization control enables sensing ‘by-eye’ of refractive index changes below 1 × 10^-3 RIU.

Determining the specific spatial distributions of elements within compound samples is of critical importance to a range of applied research fields. The usual approaches to obtaining elemental contrast involve measurement of the characteristic peaks associated with x-ray fluorescence or measuring the x-ray transmission as a function of energy. In the laboratory these measurements are challenging due to the polychromaticity and lack of tunability of the source. Here we demonstrate how newly developed, high-resolution, energy-discriminating area detector technology can be exploited to enhance elemental contrast. The detector we employ here is the Pixirad area detector which can simultaneously have up to four separate colour channels. We also discuss the potential of this new technology in the context of tomographic imaging of soft tissue.

To examine the process of endothelial cell aging we utilised hyperspectral imaging to collect broad autofluorescence emission at the individual cellular level and mathematically isolate the characteristic spectra of nicotinamide and flavin adenine dinucleotides (NADH and FAD, respectively). Quantitative analysis of this data provides the basis for a non-destructive spatial imaging method for cells and tissue. FAD and NADH are important factors in cellular metabolism and have been shown to be involved with the redox state of the cell; with the ratio between the two providing the basis for an ‘optical redox ratio’.

The advancement of biocompatible nanoplatforms with dual functionalities of diagnosis and therapeutics is strongly demanded in biomedicine in recent years. In this work, we report the synthesis and characterization of polydopamine (pD)-coated gold nanostars (Au NSs) for computed tomography (CT) imaging and enhanced photothermal therapy (PTT) of tumors. Au NSs were firstly formed via a seed-mediated growth method and then stabilized with thiolated polyethyleneimine (PEI-SH), followed by deposition of pD on their surface. The formed pD-coated Au NSs (Au-PEI@pD NSs) were well characterized. We show that the Au-PEI@pD NSs are able to convert the absorbed near-infrared laser light into heat, and have strong X-ray attenuation property. Due to the co-existence of Au NSs and the pD, the light to heat conversion efficiency of the NSs can be significantly enhanced. These very interesting properties allow their uses as a powerful theranostic nanoplatform for efficient CT imaging and enhanced phtotothermal therapy of cancer cells in vitro and the xenografted tumor model in vivo. With the easy functionalization nature enabled by the coated pD shell, the developed pD-coated Au NSs may be developed as a versatile nanoplatform for targeted CT imaging and PTT of different types of cancer.

Incomplete removal of malignant tumours continues to be a significant issue in cancer surgery. It increases the risk of local recurrence and impaired survival, and results in the need for additional surgery with associated attendant costs and morbidity. While pathological methods exist to determine tissue type during surgery, these methods can compromise post-operative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis and are restricted to excised tissue.

In this work we report the development of an optical fibre probe which could find use as an aid for margin detection during surgery. A fluorophore doped polymer coating is deposited on the tip of an optical fibre, which can then be used to record the pH by monitoring the emission spectra from the embedded indicator. The pH values of unknown tissue are measured and compared to healthy tissue, allowing for discrimination between healthy and cancerous tissue.

The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration which could find use in the operating theatre. Use of this probe in surgery either on excised or in-vivo tissue has the potential to improve success rates for complete removal of cancers.