Quantifying DNA damage following light sheet and confocal imaging of the mammalian embryo.

Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts. In this study, we employed DNA damage as a sensitive indicator of photodamage. We use light sheet microscopy with excitation at a wavelength of 405 nm for imaging embryo autofluorescence and compare its performance to laser scanning confocal microscopy. At an equivalent signal-to-noise ratio for images acquired with both modalities, light sheet microscopy reduced image acquisition time by ten-fold, and did not induce DNA damage when compared to non-imaged embryos. In contrast, imaging with confocal microscopy led to significantly higher levels of DNA damage within embryos and had a higher photobleaching rate. Light sheet imaging is also capable of inducing DNA damage within the embryo but requires multiple cycles of volumetric imaging. Collectively, this study confirms that light sheet microscopy is faster and safer than confocal microscopy for imaging live embryos, indicating its potential as a label-free diagnostic for embryo quality.

Learning algorithms for identification of whisky using portable Raman spectroscopy.

Reliable identification of high-value products such as whisky is vital due to rising issues of brand substitution and quality control in the industry. We have developed a novel framework that can perform whisky analysis directly from raw spectral data with no human intervention by integrating machine learning models with a portable Raman device. We demonstrate that machine learning models can achieve over 99% accuracy in brand or product identification across twenty-eight commercial samples. To demonstrate the flexibility of this approach, we utilized the same algorithms to quantify ethanol concentrations, as well as measuring methanol levels in spiked whisky samples. To demonstrate the potential use of these algorithms in a real-world environment we tested our algorithms on spectral measurements performed through the original whisky bottle. Through the bottle measurements are facilitated by a beam geometry hitherto not applied to whisky brand identification in conjunction with machine learning. Removing the need for decanting greatly enhances the practicality and commercial potential of this technique, enabling its use in detecting counterfeit or adulterated spirits and other high-value liquids. The techniques established in this paper aim to function as a rapid and non-destructive initial screening mechanism for detecting falsified and tampered spirits, complementing more comprehensive and stringent analytical methods.

UVA Hyperspectral Light-Sheet Microscopy for Volumetric Metabolic Imaging: Application to Preimplantation Embryo Development.

Cellular metabolism is a key regulator of energetics, cell growth, regeneration, and homeostasis. Spatially mapping the heterogeneity of cellular metabolic activity is of great importance for unraveling the overall cell and tissue health. In this regard, imaging the endogenous metabolic cofactors, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), with subcellular resolution and in a noninvasive manner would be useful to determine tissue and cell viability in a clinical environment, but practical use is limited by current imaging techniques. In this paper, we demonstrate the use of phasor-based hyperspectral light-sheet (HS-LS) microscopy using a single UVA excitation wavelength as a route to mapping metabolism in three dimensions. We show that excitation solely at a UVA wavelength of 375 nm can simultaneously excite NAD(P)H and FAD autofluorescence, while their relative contributions can be readily quantified using a hardware-based spectral phasor analysis. We demonstrate the potential of our HS-LS system by capturing dynamic changes in metabolic activity during preimplantation embryo development. To validate our approach, we delineate metabolic changes during preimplantation embryo development from volumetric maps of metabolic activity. Importantly, our approach overcomes the need for multiple excitation wavelengths, two-photon imaging, or significant postprocessing of data, paving the way toward clinical translation, such as in situ, noninvasive assessment of embryo viability.

Mitigating stimulated Brillouin scattering in multimode fibers with focused output via wavefront shaping.

The key challenge for high-power delivery through optical fibers is overcoming nonlinear optical effects. To keep a smooth output beam, most techniques for mitigating optical nonlinearities are restricted to single-mode fibers. Moving out of the single-mode paradigm, we show experimentally that wavefront-shaping of coherent input light to a highly multimode fiber can increase the power threshold for stimulated Brillouin scattering (SBS) by an order of magnitude, whilst simultaneously controlling the output beam profile. The SBS suppression results from an effective broadening of the Brillouin spectrum under multimode excitation, without broadening of transmitted light. Strongest suppression is achieved with selective mode excitation that gives the broadest Brillouin spectrum. Our method is efficient, robust, and applicable to continuous waves and pulses. This work points toward a promising route for mitigating detrimental nonlinear effects in optical fibers, enabling further power scaling of high-power fiber systems for applications to directed energy, remote sensing, and gravitational-wave detection.

Multimodal Raman spectroscopy and optical coherence tomography for biomedical analysis.

Optical techniques hold great potential to detect and monitor disease states as they are a fast, non-invasive toolkit. Raman spectroscopy (RS) in particular is a powerful label-free method capable of quantifying the biomolecular content of tissues. Still, spontaneous Raman scattering lacks information about tissue morphology due to its inability to rapidly assess a large field of view. Optical Coherence Tomography (OCT) is an interferometric optical method capable of fast, depth-resolved imaging of tissue morphology, but lacks detailed molecular contrast. In many cases, pairing label-free techniques into multimodal systems allows for a more diverse field of applications. Integrating RS and OCT into a single instrument allows for both structural imaging and biochemical interrogation of tissues and therefore offers a more comprehensive means for clinical diagnosis. This review summarizes the efforts made to date toward combining spontaneous RS-OCT instrumentation for biomedical analysis, including insights into primary design considerations and data interpretation.

The effect of discrete wavelengths of visible light on the developing murine embryo.

PURPOSE: A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. METHODS: Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. RESULTS: Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. CONCLUSION: Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.

Tailored Multi-Color Dispersive Wave Formation in Quasi-Phase-Matched Exposed Core Fibers.

Widely wavelength-tunable femtosecond light sources in a compact, robust footprint play a central role in many prolific research fields and technologies, including medical diagnostics, biophotonics, and metrology. Fiber lasers are on the verge in the development of such sources, yet widespan spectral tunability of femtosecond pulses remains a pivotal challenge. Dispersive wave generation, also known as Cherenkov radiation, offers untapped potentials to serve these demands. In this work, the concept of quasi-phase matching for multi-order dispersive wave formation with record-high spectral fidelity and femtosecond durations is exploited in selected, partially conventionally unreachable spectral regions. Versatile patterned sputtering is utilized to realize height-modulated high-index nano-films on exposed fiber cores to alter fiber dispersion to an unprecedented degree through spatially localized, induced resonances. Nonlinear optical experiments and simulations, as well as phase-mismatching considerations based on an effective dispersion, confirm the conversion process and reveal unique emission features, such as almost power-independent wavelength stability and femtosecond duration. This resonance-empowered approach is applicable to both fiber and on-chip photonic systems and paves the way to instrumentalize dispersive wave generation as a unique tool for efficient, coherent femtosecond multi-frequency conversion for applications in areas such as bioanalytics, life science, quantum technology, or metrology.

Protein detection enabled using functionalised silk-binding peptides on a silk-coated optical fibre.

We present a new coating procedure to prepare optical fibre sensors suitable for use with protein analytes. We demonstrate this through the detection of AlexaFluor-532 tagged streptavidin by its binding to D-biotin that is functionalised onto an optical fibre, via incorporation in a silk fibroin fibre coating. The D-biotin was covalently attached to a silk-binding peptide to provide SBP-biotin, which adheres the D-biotin to the silk-coated fibre tip. These optical fibre probes were prepared by two methods. The first involves dip-coating the fibre tip into a mixture of silk fibroin and SBP-biotin, which distributes the SBP-biotin throughout the silk coating (method A). The second method uses two steps, where the fibre is first dip-coated in silk only, then SBP-biotin added in a second dip-coating step. This isolates SBP-biotin to the outer surface of the silk layer (method B). A series of fluorescence measurements revealed that only the surface bound SBP-biotin detects streptavidin with a detection limit of 15 µg mL-1. The fibre coatings are stable to repeated washing and long-term exposure to water. Formation of silk coatings on fibres using commercial aqueous silk fibroin was found to be inhibited by a lithium concentration of 200 ppm, as determined by atomic absorption spectroscopy. This was reduced to less than 20 ppm by dialysis against water, and was found to successfully form a coating on optical fibres.

Scalable Functionalization of Optical Fibers Using Atomically Thin Semiconductors.

Atomically thin transition metal dichalcogenides are highly promising for integrated optoelectronic and photonic systems due to their exciton-driven linear and nonlinear interactions with light. Integrating them into optical fibers yields novel opportunities in optical communication, remote sensing, and all-fiber optoelectronics. However, the scalable and reproducible deposition of high-quality monolayers on optical fibers is a challenge. Here, the chemical vapor deposition of monolayer MoS2 and WS2 crystals on the core of microstructured exposed-core optical fibers and their interaction with the fibers' guided modes are reported. Two distinct application possibilities of 2D-functionalized waveguides to exemplify their potential are demonstrated. First, the excitonic 2D material photoluminescence is simultaneously excited and collected with the fiber modes, opening a novel route to remote sensing. Then it is shown that third-harmonic generation is modified by the highly localized nonlinear polarization of the monolayers, yielding a new avenue to tailor nonlinear optical processes in fibers. It is anticipated that the results may lead to significant advances in optical-fiber-based technologies.

All-fiber all-optical quantitative polymerase chain reaction (qPCR).

Quantitative polymerase chain reaction (qPCR), the real-time amplification and measurement of a targeted DNA molecule, has revolutionized the biological sciences and is routinely applied in areas such as medical diagnostics, forensics, and agriculture. Despite widescale use of qPCR technology in the lab, the availability of low-cost and high-speed portable systems remains one of the barriers to routine in-field implementation. Here we propose and demonstrate a potential solution using a photonics-based qPCR system. By using an all-optical approach, we achieve ultra-fast temperature response with real-time temperature feedback using nanoliter scale reaction volumes. The system uses a microcavity to act as a nanoliter scale reaction vessel with a laser-driven and optically monitored temperature cycling system for ultrafast thermal cycling and incorporates an all-fiber fluorescence excitation/detection system to achieve real-time, high sensitivity fluorescence monitoring of the qPCR process. Further, we demonstrate the potential of the system to operate as a label-free qPCR system through direct optical measurement of the sample refractive index. Due to advantages in portability and fabrication simplicity, we anticipate that this platform technology will offer a new strategy for fundamental techniques in biochemistry applications, such as point-of-care and remote diagnostics.

Single-peak fiber Bragg gratings in suspended-core optical fibers.

Femtosecond laser inscribed fiber Bragg gratings in pure-silica suspended-core optical fibers have previously been demonstrated as a promising platform for high temperature sensing. However, the density of gratings that could be written on a single fiber was limited by undesired reflections associated with higher order modes in these high numerical aperture fibers. This resulted in a complex, broadband reflection spectrum with limited multiplexing capability. In this work we utilize modifications to the fine structure of the suspended core optical fibers to fine tune the relative confinement loss of the optical fiber modes, thus reducing the contribution from such higher order modes. The effects of these changes on mode propagation are modeled, giving a range of fibers with different confinement loss properties which can be tailored to the specific length scale of a desired application. We achieve single-peak reflections from individual fiber Bragg gratings, significantly improving performance for multipoint sensing and demonstrate this technique by writing 20 gratings onto a single fiber.

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus-oocyte-complex (COC).

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non-invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 µL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell-safe with no inherent cell toxicity or light-induced phototoxicity indicated by negative DNA damage staining. Pre-measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01-0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

Minocycline attenuates 3,4-methylenedioxymethamphetamine-induced hyperthermia in the rat brain.

Hyperthermia is most dangerous clinical symptom of acute MDMA administration, and a key factor related to potentially life-threatening MDMA-induced complications. MDMA induces a consistently faster onset of brain hyperthermia when compared to a delayed and moderate hyperthermia in the body, and the most harmful effects of MDMA are related to its modulation of neural functions. The primary focus of this study was to investigate the effects of minocycline, a centrally acting tetracycline derivative on MDMA-induced brain hyperthermia at high ambient temperature. However, we also simultaneously recorded body temperature, heart rate, and locomotor activity changes, allowing us to gain a better understanding of the mechanisms underlying the MDMA-induced hyperthermic response. We also investigated the effects of MDMA at normal ambient temperature to provide further evidence as to the importance of environmental factors on the intensity of MDMA's temperature effects. At normal ambient temperature, MDMA (10 mg/kg, i.p.) induced a significant brain and body hypothermia for the first 90 min following drug administration, and significantly increased heart rate and locomotor activity compared to saline controls. At high ambient temperature however, MDMA (10 mg/kg, i.p.) induced a robust and extended brain and body hyperthermia, as well as significantly increased heart rate and locomotor activity. A 3-day minocycline (50 mg/kg, i.p.) pre-treatment significantly attenuated MDMA-induced increases in brain temperature, body temperature, heart rate, and locomotor activity. Our findings indicate that minocycline is more effective in attenuating the exacerbated MDMA-induced hyperthermic response in the brain compared to the body at high ambient temperature.

Quantum noise limited nanoparticle detection with exposed-core fiber.

Label-free biosensors are important tools for clinical diagnostics and for studying biology at the single molecule level. The development of optical label-free sensors has allowed extreme sensitivity but can expose the biological sample to photodamage. Moreover, the fragility and complexity of these sensors can be prohibitive to applications. To overcome these problems, we develop a quantum noise limited exposed-core fiber sensor providing robust platform for label-free biosensing with a natural path toward microfluidic integration. We demonstrate the detection of single nanoparticles down to 25 nm in radius with optical intensities beneath known biophysical damage thresholds.

Tunable multi-wavelength third-harmonic generation using exposed-core microstructured optical fiber.

We demonstrate that exposed-core microstructured optical fibers offer multiple degrees of freedom for tailoring third-harmonic generation through the core diameter, input polarization, and nanofilm deposition. Varying these parameters allows control of the phase-matching position between an infrared pump wavelength and the generated visible wavelengths. In this Letter, we show how increasing the core diameter over previous experiments (2.57 µm compared to 1.85 µm) allows the generation of multiple wavelengths, which can be further controlled by rotating the input pump polarization and the deposition of dielectric nanofilms. This can lead to highly tailorable light sources for applications such as spectroscopy or nonlinear microscopy.

Improved method for optical fiber temperature probe implantation in brains of free-moving rats.

BACKGROUND: The localized monitoring of brain temperature is crucial to the understanding of the mechanisms underlying brain hyperthermia, such as that caused by stimulant drugs. Many animal studies investigating brain hyperthermia have utilized thermocouple electrodes for temperature measurement, however optical fiber sensors have proven to be an attractive alternative to conventional measurement techniques. Despite their advantages, optical fiber sensors in their current form have struggled to find effective use in studies involving free-moving animals. NEW METHOD: We have developed an improved optical fiber temperature probe and implantation method suitable for sensing in free-moving animals. By altering the structure of the probe, conventional guide cannulae can be used for stereotaxic implantation thus increasing ease-of-use and probe durability. RESULTS: The new probe structure was easily implanted and extremely durable both pre-experimentation and during sampling in vivo. Probe re-usability also allowed for increased experimental workflow. Rats administered MDMA showed pathological increases in brain temperature. COMPARISON WITH EXISTING METHOD(S): Thermocouples commonly used for temperature measurement in deep brain structures lack the advantages offered by optical fiber sensors. Unlike our improved design, previous optical fiber temperature probes were unable to be removed from the brains of rats without removing the dental cement affixing it to the skull. This made the probe susceptible to breakage and often resulted in the complete loss of the animal from the experiment. CONCLUSIONS: Our fiber temperature probe and revised implantation technique can be easily employed in brain thermorecording using advantageous optical fiber sensors suitable for use in awake free-moving animals.

Miniaturized single-fiber-based needle probe for combined imaging and sensing in deep tissue.

The ability to visualize structure while simultaneously measuring chemical or physical properties of a biological tissue has the potential to improve our understanding of complex biological processes. We report the first miniaturized single-fiber-based imaging+sensing probe capable of simultaneous optical coherence tomography (OCT) imaging and temperature sensing. An OCT lens is fabricated at the distal end of a double-clad fiber, including a thin layer of rare-earth-doped tellurite glass to enable temperature measurements. The high refractive index of the tellurite glass enables a common-path interferometer configuration for OCT, allowing easy exchange of probes for biomedical applications. The simultaneous imaging+sensing capability is demonstrated on rat brains.

Plasmonic nanoparticle-functionalized exposed-core fiber-an optofluidic refractive index sensing platform.

Here, we show that immobilizing ensembles of gold nanospheres within tailored areas on the open side of an exposed-core microstructured fiber yields a monolithic, highly sensitive plasmon-based refractive index sensor. The nanoparticle densities (average nanoparticle diameter: 45 nm) on the small-core fiber (core diameter: 2.5 µm) are controlled electrostatically, yielding densities of 4 nanoparticles/µm2. Refractive index sensitivities of 200 nm/RIU for aqueous analytes at high fringe contrast levels (-20 dB) have been observed. Our concept presents an easy-to-use, efficient, and multiplex-compatible sensing platform for rapid small-volume detection with the capacity for integration into a bioanalytic, optofluidic, or microfluidic system.

Cancer Detection in Human Tissue Samples Using a Fiber-Tip pH Probe.

Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative 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 fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. 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 theater. 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. Cancer Res; 76(23); 6795-801. ©2016 AACR.

Quantification of the fluorescence sensing performance of microstructured optical fibers compared to multi-mode fiber tips.

Microstructured optical fibers, particularly those with a suspended-core geometry, have frequently been argued as efficient evanescent-field fluorescence-based sensors. However, to date there has not been a systematic comparison between such fibers and the more common geometry of a multi-mode fiber tip sensor. In this paper we make a direct comparison between these two fiber sensor geometries both theoretically and experimentally. Our results confirm that suspended-core fibers provide a significant advantage in terms of total collected fluorescence signal compared to multi-mode fibers using an equivalent experimental configuration.