Integrating Cavity Enhanced Spectroscopy for Liquid and Gas Sensing

With continued advances in optical instrumentation coupled with new highly specific contrast methods, optical based spectroscopic techniques continue to find new applications in a wide range of fields from medical diagnostics to analytical chemistry. Fluorescence based detection has been used in a variety of medical and environmental sensing applications. Raman spectroscopy has long been used in a variety of industries and applications due to its unique ability to provide label free chemically specific information about a molecule. While both of these techniques have tremendous potential in both medical diagnostics and environmental sensing, often cost efficiency and sensitivity limit more wide spread use. To overcome these limitations, we explore the use of integrating cavity enhanced spectroscopy as a means to greatly enhance the detection sensitivity of both Raman and fluorescence spectroscopy based detection. Using a new diffuse reflector, we have constructed novel integrating cavities that provide significant enhancement of linear optical processes. Enhancement of these processes stems from the long optical pathlength light experiences inside the cavity combined with the relatively large volume that can be probed when compared to traditional spectroscopic approaches. Integrating cavities have several advantages over other sources of signal enhancement in that there is no need for high cost laser sources, and the broad range of wavelengths over which the cavity is reflective affords the ability to use ultraviolet (UV) and blue excitation wavelengths. Raman spectroscopy greatly benefits from the use of more blue shifted sources. Additionally, this allows for one to take advantage of the endogenous fluorescence of many organic molecules that fluoresce under UV excitation. The enhancement of both spontaneous Raman generation as a result of the integrating cavity is demonstrated by measuring the sensitivity of detection for three aromatic hydrocarbons. Fluorescence enhancement is demonstrated by exploring the detection limits of an urobilin zinc phosphor complex, a biomarker that has been shown to be a viable indicator of human and animal waste contamination in water sources. Finally, the design of an integrating cavity system for gas phase analysis will be demonstrated

Stobe Photography Mapping of Cell Membrane Potential with Nanosecond Resolution

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns–10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15–20% higher LD50, p \u3c 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30–40% lower LD50 (p \u3c 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation

Control of the Electroporation Efficiency of Nanosecond Pulses by Swinging the Electric Field Vector Direction

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles \u3c160° (1 Hz) and \u3c130° (833 kHz). Facilitation plateaued at about three-fold stronger electroporation compared to single pulses at 90–100° angle for both nsEP frequencies. The profound dependence of the efficiency on the angle enables novel protocols for highly selective focal electroporation at one electrode in a three-electrode array while avoiding effects at the other electrodes. Nanosecond-resolution imaging of cell membrane potential was used to link the selectivity to charging kinetics by co- and counter-directional nsEPs

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU

Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy

Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment

Bright emission from a random Raman laser

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm(−1). Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium