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Burns are thermal injuries that can completely damage or at least compromise the protective function of skin, and affect the ability of tissues to manage moisture. Burn-damaged tissues exhibit lower elasticity than healthy tissues, due to significantly reduced water concentrations and plasma retention. Current methods for determining burn intensity are limited to visual inspection, and potential hospital x-ray examination. We present a unique confocal microscope capable of measuring Raman and Brillouin spectra simultaneously, with concurrent fluorescence investigation from a single spatial location, and demonstrate application by investigating and characterizing the properties of burn-afflicted tissue on chicken skin model. Raman and Brillouin scattering offer complementary information about a material's chemical and mechanical structure, while fluorescence can serve as a useful diagnostic indicator and imaging tool. The developed instrument has the potential for very diverse analytical applications in basic biomedical science and biomedical diagnostics and imaging.

Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique microscopy system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 50kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.

Nanosecond pulsed electric fields (nsPEF) have proven useful for transporting cargo across cell membranes and selectively activating cellular pathways. The chemistry and biophysics governing this cellular response, however, are complex and not well understood. Recent studies have shown that the conductivity of the solution cells are exposed in could play a significant role in plasma membrane permeabilization and, thus, the overall cellular response. Unfortunately, the means of detecting this membrane perturbation has traditionally been limited to analyzing one possible consequence of the exposure – diffusion of molecules across the membrane. This method has led to contradictory results with respect to the relationship between permeabilization and conductivity. Diffusion experiments also suffer from “saturation conditions” making multi-pulse experiments difficult. As a result, this method has been identified as a key stumbling block to understanding the effects of nsPEF exposure. To overcome these limitations, we recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) that allows us to identify nanoporation in live cells during the pulse in a wide array of conditions. As a result, we are able to explore and fully test whether lower conductivity extracellular solutions could induce more efficient nanoporation. This hypothesis is based on membrane charging and the relative difference between the extracellular solution and the cytoplasm. The experiments also allow us to test the noise floor of our methodology against the effects of ion leakage. The results emphasize that the electric field, not ionic phenomenon, are the driving force behind nsPEF-induced membrane nanoporation.

Irreversible electroporation therapy is utilized to remove cancerous tissues thru the delivery of rapid (250Hz) and high voltage (V) (1,500V/cm) electric pulses across microsecond durations. Clinical research demonstrated that bipolar (BP) high voltage microsecond pulses opposed to monophasic waveforms relieve muscle contraction during electroporation treatment. Our group along with others discovered that nanosecond electric pulses (nsEP) can activate second messenger cascades, induce cytoskeletal rearrangement, and depending on the nsEP duration and frequency, initiate apoptotic pathways. Of high interest across in vivo and in vitro applications, is how nsEP affects muscle physiology, and if nuances exist in comparison to longer duration electroporation applications. To this end, we exposed mature skeletal muscle cells to monopolar (MP) and BP nsEP stimulation across a wide range of electric field amplitudes (1-20 kV/cm). From live confocal microscopy, we simultaneously monitored intracellular calcium dynamics along with nsEP-induced muscle movement on a single cell level. In addition, we also evaluated membrane permeability with Yo-PRO-1 and Propidium Iodide (PI) across various nsEP parameters. The results from our findings suggest that skeletal muscle calcium dynamics, and nsEP-induced contraction exhibit exclusive responses to both MP and BP nsEP exposure. Overall the results suggest in vivo nsEP application may elicit unique physiology and field applications compared to longer pulse duration electroporation.

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

Short infrared laser pulses induce a variety of effects in cells and tissues, including neural stimulation and inhibition. However, the mechanism behind these physiological effects is poorly understood. It is known that the fast thermal gradient induced by the infrared light is necessary for these biological effects. Therefore, this study tests the hypothesis that the fast thermal gradient induced in a cell by infrared light exposure causes a change in the membrane fluidity. To test this hypothesis, we used the membrane fluidity dye, di-4-ANEPPDHQ, to investigate membrane fluidity changes following infrared light exposure. Di-4-ANEPPDHQ fluorescence was imaged on a wide-field fluorescence imaging system with dual channel emission detection. The dual channel imaging allowed imaging of emitted fluorescence at wavelengths longer and shorter than 647 nm for ratiometric assessment and computation of a membrane generalized polarization (GP) value. Results in CHO cells show increased membrane fluidity with infrared light pulse exposure and this increased fluidity scales with infrared irradiance. Full recovery of pre-infrared exposure membrane fluidity was observed. Altogether, these results demonstrate that infrared light induces a thermal gradient in cells that changes membrane fluidity.

Short infrared laser pulses have many physiological effects on cells including the ability to stimulate action potentials in neurons. Here we show that short infrared laser pulses can also reversibly block action potentials. Primary rat hippocampal neurons were transfected with the Optopatch2 plasmid, which contains both a blue-light activated channel rhodopsin (CheRiff) and a red-light fluorescent membrane voltage reporter (QuasAr2). This optogenetic platform allows robust stimulation and recording of action potential activity in neurons in a non-contact, low noise manner. For all experiments, QuasAr2 was imaged continuously on a wide-field fluorescent microscope using a Krypton laser (647 nm) as the excitation source and an EMCCD camera operating at 1000 Hz to collect emitted fluorescence. A co-aligned Argon laser (488 nm, 5 ms at 10Hz) provided activation light for CheRiff. A 200 mm fiber delivered infrared light locally to the target neuron. Reversible action potential block in neurons was observed following a short infrared laser pulse (0.26-0.96 J/cm2; 1.37-5.01 ms; 1869 nm), with the block persisting for more than 1 s with exposures greater than 0.69 J/cm2. Action potential block was sustained for 30 s with the short infrared laser pulsed at 1-7 Hz. Full recovery of neuronal activity was observed 5-30s post-infrared exposure. These results indicate that optogenetics provides a robust platform for the study of action potential block and that short infrared laser pulses can be used for non-contact, reversible action potential block.

We recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) to identify membrane disruption events in live cells. This technique was used to detect nanoporation in the plasma membrane following nanosecond pulsed electric field (nsPEF) exposure. It has been hypothesized that similar poration events could be induced by the thermal gradients generated by infrared (IR) laser energy. Optical pulses are a highly desirable stimulus for the nervous system, as they are capable of inhibiting and producing action potentials in a highly localized but non-contact fashion. However, the underlying mechanisms involved with infrared neural stimulation (INS) are not well understood. The ability of our method to non-invasively measure membrane structure and transmembrane potential via Two Photon Fluorescence (TPF) make it uniquely suited to neurological research. In this work, we leverage our technique to understand what role membrane structure plays during INS and contrast it with nsPEF stimulation. We begin by examining the effect of IR pulses on CHO-K1 cells before progressing to primary hippocampal neurons. The use of these two cell lines allows us to directly compare poration as a result of IR pulses to nsPEF exposure in both a neuron-derived cell line, and one likely lacking native channels sensitive to thermal stimuli.

Cellular delivery of pulsed IR laser energy has been shown to stimulate action potentials in neurons. The mechanism for this stimulation is not completely understood. Certain hypotheses suggest the rise in temperature from IR exposure could activate temperature- or pressure-sensitive channels, or create pores in the cellular outer membrane. Studies using intensity-based Ca 2+- responsive dyes show changes in Ca 2+ levels after various IR stimulation parameters; however, determination of the origin of this signal proved difficult. An influx of larger, typically plasma-membrane-impermeant ions has been demonstrated, which suggests that Ca 2+ may originate from the external solution. However, activation of intracellular signaling pathways, possibly indicating a more complex role of increasing Ca 2+ concentration, has also been shown. By usingCa 2+ sensitive dye Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the excitation wavelengths, we have quantified the Ca 2+ mobilization in terms of influx from the external solution and efflux from intracellular organelles. CHO-K1 cells, which lack voltage-gated Ca 2+ channels, and NG-108 neuroblastoma cells, which do not produce action potentials in an early undifferentiated state, are used to determine the origin of the Ca 2+ signals and investigate the role these mechanisms may play in IR neural stimulation.

Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell’s ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line’s higher tolerance of nsPEF exposure.

Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to or ientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di-4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.

Despite 30 years of research, the mechanism behind the induced breakdown of plasma membranes by electrical pulses, termed electroporation, remains unknown. Current theories treat the interaction between the electrical field and the membrane as an entirely electrical event pointing to multiple plausible mechanisms. By investigating the biophysical interaction between plasma membranes and nanosecond electrical pulses (nsEP), we may have identified a non-electric field driven mechanism, previously unstudied in nsEP, which could be responsible for nanoporation of plasma membranes. In this investigation, we use a non-contact optical technique, termed probe beam deflection technique (PBDT), to characterize acoustic shockwaves generated by nsEP traveling through tungsten wire electrodes. We conclude these acoustic shockwaves are the result of the nsEP exposure imparting electrohydraulic forces on the buffer solution. When these acoustic shockwaves occur in close proximity to lipid bilayer membranes, it is possible that they impart a sufficient amount of mechanical stress to cause poration of that membrane. This research establishes for the first time that nsEP discharged in an aqueous medium generate measureable pressure waves of a magnitude capable of mechanical deformation and possibly damage to plasma membranes. These findings provide a new insight into the longunanswered question of how electric fields cause the breakdown of plasma membranes.

The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular or ganelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activ ity of Jurkat and U937 cells after nsPE F. The XF Analyzer uses a transient micro-chamber of only a few µL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in bo th U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects. Keywords : nanosecond pulse electric field, mitochondria, Seahorse, mitochondrial function basal respiration, ATP production, maximal respiration, oxygen consumpti on rate (OCR), extracellular acidification rate (EAR) *Larry.e.estlack.ctr@mail.mil; phone 1 210 539-5945, fax 1 210 539-7945

It has been shown that exposure of live neurons to a low-intensity pulsed infrared light can be used to excite action potentials. Infrared pulsed laser coupled to an optical fiber can be utilized to create a rapid localized increase in temperature in the vicinity of the cell. The resulting temperature gradient leads to an increase in membrane fluidity and permeability, causing depolarization of the target cell. In order to characterize the fluidity of the cell membrane at various temperatures with and without pulsed IR light exposure, we used a polarity-sensitive fluorescent probe di-4- ANEPPDHQ. This dye exhibits a fluorescent shift between the disordered and ordered phases of the membrane, and can be used to quantitatively evaluate the state of the membrane by calculating the generalized polarization (GP) value. Using high-speed imaging of cells exposed to a IR light of varying pulse width, it was determined that a longer pulse width leads to a greater change in the GP value. Comparison of GP values of cells at different ambient temperatures without the pulsed IR light exposure and cells exposed to pulsed IR light indicated that a rapid temperature gradient caused by the exposure to pulsed light induces a larger change in GP value than the ambient temperature increase alone, indicating a greater disruption of membrane fluidity and permeability.

Previously, it was demonstrated that small nanometer-sized pores (nanopores) are preferentially formed after exposure to nanosecond pulsed electric fields (nsPEF). We have reported that nanoporation of the plasma membrane directly affects the phospholipids of the cell membrane, ultimately culminating in phosphatidylinositol 4,5 - bisphosphate (PIP 2 ) intracellular signaling. PIP 2 , located within the internal layer of the plasma membrane, plays a critical role as a regulator of ion transport proteins, a source of second messenger compounds, and an anchor for cytoskeletal elements. In this proceeding, we present data that demonstrates that nsPEFs initiate electric field dose-dependent PIP 2 hydrolysis and/or depletion from the plasma membrane through the observation of the accumulation of inositol 1,4,5 -trisphosphate (IP3) in the cytoplasm and the increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. The phosphoinositide signaling cascade presented here involves activation of phospholipase C (PLC) and protein kinase C (PKC), which are responsible for a multitude of biological effects after nsPEF exposure. These results expand our current knowledge of nsPEF induced physiological effects, and serve as a basis for development of novel tools for drug independent stimulation or modulation of different cellular functions.

Nanosecond pulsed electric fields (nsPEFs) are known to increase cell membrane permeability to small molecules in accordance with dosages. As previous work has focused on nsPEF exposures in whole cells, electrodeformation may contribute to this induced-permeabilization in addition to other biological mechanisms. Here, we hypothesize that cellular elasticity, based upon the cytoskeleton, affects nsPEF-induced decrease in cellular viability. Young’s moduli of various types of cells have been calculated from atomic force microscopy (AFM) force curve data, showing that CHO cells are stiffer than non-adherent U937 and Jurkat cells, which are more susceptible to nsPEF exposure. To distinguish any cytoskeletal foundation for these observations, various cytoskeletal reagents were applied. Inhibiting actin polymerization significantly decreased membrane integrity, as determined by relative propidium uptake and phosphatidylserine externalization, upon exposure at 150 kV/cm with 100 pulses of 10 ns pulse width. Exposure in the presence of other drugs resulted in insignificant changes in membrane integrity and 24-hour viability. However, Jurkat cells showed greater lethality than latrunculin-treated CHO cells of comparable elasticity. From these results, it is postulated that cellular elasticity rooted in actin-membrane interaction is only a minor contributor to the differing responses of adherent and non-adherent cells to nsPEF insults.

Exposure to nano-second pulsed electrical fields (nsPEFs) has been shown to cause poration of external and internal cell membranes, DNA damage, and blebbing of the plasma membrane. Recovery from nsPEF exposure is likely dependent on multiple factors, including exposure parameters, length of time between pulses, and extent of cellular damage. As cells progress through the cell cycle, variations in DNA and nucleus structure, cytoskeletal arrangement, and elasticity of cell membrane could cause nsPEFs to affect cells differently during different cell cycle phases. To better understand the impact of nsPEF on cell cycle, we investigated CHO cell cycle progression following varying intensities of nsPEFexposures. Cell populations were examined post exposure (10 ns pulse trains at 100, 150, or 200kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis to determine cell cycle phase. Populations exhibited arrest in G2/M phase, but not in G1 phase at 1h post-exposure that increased in severity and duration with increasing exposure dose. Recovery from arrest was complete after 12h, and populations did not exhibit an increase in apoptosis as a result of exposure. Post exposure arrest in G2/M phase may indicate that nsPEF-induced damage is not significant to cause G1 arrest or that mitotic checkpoints are more important regulators of cell cycle progression after nsPEF exposure.

The exposure of nanosecond pulsed electric fields (nsPEF) to living cells has been shown to create nanopores in the plasma membranes. These nanopores allow the passage of small ions but exclude the transport of larger molecules such as Propidium ions, with permeabilization persisting for many minutes. To characterize these nanopores and the effect of temperature of the formation and resealing of these pores, we have chosen to use 6-Propionyl-2-(N,NDimethylamo) Naphthalene (PRODAN) as an indicator of membrane organization. PRODAN is a fluorescent dye with a large excited-state dipole moment that displays extensive solvent polarity-dependent fluorescent shifts. By monitoring this shift in fluorescence spectrum, disruption of the membrane after an electric exposure is observed as an immediate increase in the membrane fluidity, likely indicating poration of the membrane. High-speed imaging results indicate that a change in membrane organization occurs instantly (

Terahertz time-domain spectroscopy (THz-TDS) is an effective technique to probe the intermolecular and collective vibrational modes of biological macromolecules at THz frequencies. To date, the vast majority of spectroscopic studies have been performed on dehydrated biomolecular samples. Given the fact that all biochemical processes occur in aqueous environments and water is required for proper protein folding and function, we hypothesize that valuable information can be gained from spectroscopic studies performed on hydrated biomolecules in their native conformation. In this study, we used a THz-TDS system that exploits photoconductive techniques for THz pulse generation and freespace electro-optical sampling approaches for detection. We used the THz spectrometer to measure the time-dependent electric field of THz waves upon interaction with water, phosphate buffered saline (PBS), and collagen gels. By comparing these waveforms with references, we simultaneously determined each sample's index of refraction (n) and absorption coefficients (μa) as a function of frequency. Our data show that the properties we measure for the water, PBS and collagen are comparable to those reported in the literature. In the future, we plan to examine the effect that both temperature and pH have on the optical properties of other biological macromolecules. Studies will also be performed to compare our results to those generated using molecular dynamics simulations.

Cellular exposure to nanosecond pulsed electric fields (nsPEF) are believed to cause immediate creation of nanopores in the plasma membrane. These nanopores enable passage of small ions, but remain impermeable to larger molecules like propidium iodide. Previous work has shown that nanopores are stable for minutes after exposure, suggesting that formation of nanopores in excitable cells could lead to prolonged action potential inhibition. Previously, we measured the formation of nanopores in neuroblastoma cells by measuring the influx of extracellular calcium by preloading cells with Calcium Green-AM. In this work, we explored the impact of changing the width of a single nsPEF, at constant amplitude, on uptake of extracellular calcium ions by primary hippocampal neurons (PHN). Calcium Green was again used to measure the influx of extracellular calcium and FM1-43 was used to monitor changes in membrane conformation. The observed thresholds for nanopore formation in PHN by nsPEF were comparable to those measured in neuroblastoma. This work is the first study of nsPEF effects on PHN and strongly suggests that neurological inhibition by nanosecond electrical pulses is highly likely at doses well below irreversible damage.

Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

Ultrashort or nanosecond electrical pulses (USEP) cause repairable damage to the plasma membranes of cells through formation of nanopores. These nanopores are able to pass small ions such as sodium, calcium, and potassium, but remain impermeable to larger molecules like trypan blue and propidium iodide. What remains uncertain is whether generation of nanopores by ultrashort electrical pulses can inhibit action potentials in excitable cells. In this paper, we explored the sensitivity of excitable cells to USEP using Calcium Green AM 1 ester fluorescence to measure calcium uptake indicative of nanopore formation in the plasma membrane. We determined the threshold for nanopore formation in neuroblastoma cells for three pulse parameters (amplitude, pulse width, and pulse number). Measurement of such thresholds will guide future studies to determine if USEP can inhibit action potentials without causing irreversible membrane damage.

The cellular response to subtle membrane damage following exposure to nanosecond electric pulses (nsEP) is not well understood. Recent work has shown that when cells are exposed to nsEP, ion permeable nanopores (< 2nm) are created in the plasma membrane in contrast to larger diameter pores (> 2nm) created by longer micro and millisecond duration pulses. Macroscopic damage to a plasma membrane by a micropipette has been shown to cause internal vesicles (lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis. Formation of large pores in the plasma membrane by electrical pulses has been shown to elicit lysosomal exocytosis in a variety of cell types. Our research objective is to determine whether lysosomal exocytosis will occur in response to nanopores formed by exposure to nsEP. In this paper we used propidium iodide (PI) and Calcium Green-1 AM ester (CaGr) to differentiate between large and small pores formed in CHO-K1 cells following exposure to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm. This information was compared to changes in membrane organization observed by increases in FM1-43 fluorescence, both in the presence and absence of calcium ions in the outside buffer. In addition, we monitored the real time migration of lysosomes within the cell using Cellular Lights assay to tag LAMP-1, a lysosomal membrane protein. Both 1 and 20 pulses elicited a large influx of extracellular calcium, while little PI uptake was observed following a single pulse exposure. Statistically significant increases in FM1-43 fluorescence were seen in samples containing calcium suggesting that calcium-triggered membrane repair may be occurring. Lastly, density of lysosomes within cells, specifically around the nucleus, appeared to change rapidly upon nsEP stimulation suggesting lysosomal migration.

Finite-difference time-domain (FDTD) methods are widely used to model the propagation of electromagnetic radiation in biological tissues. High-performance central processing units (CPUs) can execute FDTD simulations for complex problems using 3-D geometries and heterogeneous tissue material properties. However, when FDTD simulations are employed at terahertz (THz) frequencies excessively long processing times are required to account for finer resolution voxels and larger computational modeling domains. In this study, we developed and tested the performance of 2-D and 3-D FDTD thermal propagation code executed on a graphics processing unit (GPU) device, which was coded using an extension of the C language referred to as CUDA. In order to examine the speedup provided by GPUs, we compared the performance (speed, accuracy) for simulations executed on a GPU (Tesla C2050), a high-performance CPU (Intel Xeon 5504), and supercomputer. Simulations were conducted to model the propagation and thermal deposition of THz radiation in biological materials for several in vitro and in vivo THz exposure scenarios. For both the 2-D and 3-D in vitro simulations, we found that the GPU performed 100 times faster than runs executed on a CPU, and maintained comparable accuracy to that provided by the supercomputer. For the in vivo tissue damage studies, we found that the GPU executed simulations 87x times faster than the CPU. Interestingly, for all exposure duration tested, the CPU, GPU, and supercomputer provided comparable predictions for tissue damage thresholds (ED 50 ). Overall, these results suggest that GPUs can provide performance comparable to a supercomputer and at speeds significantly faster than those possible with a CPU. Therefore, GPUs are an affordable tool for conducting accurate and fast simulations for computationally intensive modeling problems.

Intense, nanosecond-duration electric pulses (nsEP) have been introduced as a novel modality to alter cellular function, with a mechanism of action qualitatively different from micro- and millisecond duration pulses used in electroporation. In this study, we determined the thresholds for plasma membrane injury (within 15 minutes) and cell death (at 24 hours) for 4 different cell types (CHO-K1, HeLa, Jurkat and U937). Plasma membrane injury was measured by flow cytometry using two fluorescent dyes, namely Annexin V-FITC, which binds to phosphatidylserine (PS) upon its externalization (subtle membrane injury), and propidium iodide (PI), which is typically impermeable to the cell, but enters when large pores are formed in the plasma membrane. In all cell types, 10-ns pulses caused phosphatidylserine (PS) externalization at low doses (

In recent years, numerous security, military, and medical applications have been developed which use Terahertz (THz) radiation. These developments have heightened concerns in regards to the potential health risks that are associated with this type of radiation. To determine the cellular and molecular effects caused by THz radiation, we exposed several human cell lines to high-power THz radiation, and then we determined death thresholds and gene expression profiles. Necrotic and apoptotic death thresholds were determined for Jurkat cells using an optically-pumped molecular gas THz source (υ = 2.52 THz, H = 227 mW/cm 2 ), MTT viability assays, and flow cytometric techniques. In addition, we used confocal microscopic techniques to demarcate lethal spatial regions in a monolayer of dermal fibroblasts exposed to THz radiation. Then, to determine if cells exhibit a THz-specific gene expression signature, we exposed dermal fibroblasts to THz radiation and analyzed their transcriptional response using microarray gene chips. We found that 60% of the Jurkat cells survived the 30-minute THz exposure, whereas only 20% survived the 40-minute exposure. The flow data confirmed these results and provided evidence that THz-induced cell death was mediated using both nectrotic and apoptotic processes. The preliminary microscopy studies provided convincing evidence warranting future efforts using these techniques. Lastly, we found that dermal fibroblasts up-regulated several genes when exposed to THz radiation. Overall, these results provide evidence for the cellular and molecular effects associated with THz radiation, and we speculate that the identified up-regulated genes may serve as excellent candidate biomarkers for THz exposures.

Terahertz (THz) radiation is increasingly being used in biomedical imaging and spectroscopy applications. These techniques show tremendous promise to provide new sophisticated tools for the improved detection of skin cancer. However, despite recent efforts to develop these applications, few studies have been conducted to characterize the optical properties of skin at THz frequencies. Such information is required to better understand THz-tissue interactions, and is critical for determining the feasibility of proposed applications. In this study, we have developed and tested a THz time-domain spectroscopy system. We used this system to acquire the optical properties for fresh and frozen/thawed excised porcine skin from 0.1 to 2.0 THz. Results show that the index of refraction (n) for both frozen and fresh skin decreases with frequency. For frozen skin, n equals 2.5 at 0.1 THz and 2.0 at 2.0 THz, and for fresh skin equals 2.0 at 0.1 THz and 1.7 at 2.0 THz. Values for the absorption coefficient (μa) increase with frequency for both frozen and fresh skin. Frozen skin exhibits μa values equal to 56 cm-1 at 0.1 THz and 550 cm-1 at 2.0 THz, whereas fresh skin exhibits values of 56 cm-1 at 0.1 THz and 300 cm-1 at 2.0 THz. Assuming the optical penetration depth (δ) is inversely proportional to μa (absorption-dominated interactions), THz radiation has limited δ in skin (200 μm at 0.1 THz to 40 μm at 2.0 THz). These results suggest that applications exploiting THz radiation show the most promise for investigating superficial tissues.

The biological effects associated with Terahertz (THz) radiation are not well characterized. In this study, we investigated the cellular response of human dermal fibroblasts exposed to an optically-pumped molecular gas THz laser (υ = 2.52 THz, irradiance = 84.8 mW/cm 2 , exposure duration = 5 to 80 minutes). Computational dosimetry was conducted using finite-difference time-domain (FDTD) modeling techniques. Empirical dosimetry was conducted using infrared cameras and thermocouples. Cellular viability was assessed 24 h post-exposure using MTT calorimetric assays. Quantitative PCR was performed 4 h post-exposure to evaluate the transcriptional activation of genes involved in protein and DNA damage pathways. Comparable analyses were also performed for hyperthermic and genotoxic positive control samples. For all of the exposure durations tested, we found that greater than 95% of the cells were viable post-exposure. In addition, the exposed cells showed only minor increases (~3.5-fold) in heat shock protein expression. The empirical dosimetric data showed that the temperature of the cells increased by ~3 °C during exposure. This value was consistent with that predicted by the computational models. Interestingly, although the THz-exposed cells exhibited increases in heat shock protein expression, the magnitude of these increases was comparable to those observed in hyperthermic controls. In addition, none of the DNA repair genes tested were up-regulated in the THz-exposed cells, whereas 40-fold increases were observed in the genotoxic control cells. These results suggest that the biological effects imposed by THz radiation appear to be primarily photothermal in nature.

The terahertz (THz) region of the electromagnetic (EM) spectrum is defined as frequencies ranging from 0.1 to 10 THz. The optical properties of biological tissues have been characterized in neighboring spectral regions; however, few studies have been conducted that have examined these properties in the THz wavelength range. In this study, we used a far-infrared optically-pumped terahertz laser system, a reflection spectrometer system, and photothermal radiometric techniques to characterize the optical properties of water and biological tissues. The reflection spectrometer system performed well at lower frequencies, but proved to be unsuitable for frequencies greater than 2.52 THz. The suboptimal performance was determined to be primarily due to the higher transmission losses of the lenses, and the increased atmospheric losses that are associated with higher terahertz frequencies. The waveguide studies corroborated these findings and served to demonstrate that purging the laser beam path with nitrogen gas was an effective way to markedly reduce THz beam propagation losses. Given this finding, we have designed a temperature-controlled, nitrogen gas purged THz enclosure. The preliminary studies using photothermal radiometric techniques appeared to provide reasonable measures for the absorption coefficient (μa) of water at THz frequencies. In future studies, the tissue property measurements will made within the custom-designed enclosure using photothermal radiometric techniques.

Cellular exposure to nanosecond duration electrical pulses (nsEP) has been shown to elicit a marked decrease in plasma membrane resistance hypothesized to be due to formation of nanopores. Patch clamp technique has been used to measure changes in plasma membrane resistance immediately following nsEP exposure. Previous research by our group identified absorbed dose as a single metric to quantify cellular exposure across various pulse parameters. In that work, the relationship between the drop in membrane resistance and absorbed dose was hypothesized to be a measure of cell sensitivity to nsEP. In this study, we explore this hypothesis by employing patch clamp technique prior to exposure to measure whole cell currents at time points before and after exposure. Using two cell lines (GH3 and CHO), dose response curves were generated for single 60ns pulse exposures at 10 and 90 seconds post exposure. From this data, it was evident that dose response curves at 10 seconds post exposure showed increased permeability following nsEP exposure compared to 90 seconds post exposure. We attribute this difference to active recovery of the cell to nanoporation. However, at 90 seconds, the results for both cell types appeared to nearly match previously published results proving that a cell in whole cell configuration is slightly more sensitive to nsEP exposure. Future work will extend this study to include multiple nsEP exposures and additional cell lines.

Recently, an extensive layer of intra-cellular signals was discovered that was previously undetected by genetic radar. It is now known that this layer consists primarily of a class of short noncoding RNA species that are referred to as microRNAs (miRNAs). MiRNAs regulate protein synthesis at the post-transcriptional level, and studies have shown that they are involved in many fundamental cellular processes. In this study, we hypothesized that miRNAs may be involved in cellular stress response mechanisms, and that cells exposed to thermal stress may exhibit a signature miRNA expression profile indicative of their functional involvement in such mechanisms. To test our hypothesis, human dermal fibroblasts were exposed to an established hyperthermic protocol, and the ensuing miRNA expression levels were evaluated 4 hr post-exposure using microRNA microarray gene chips. The microarray data shows that 123 miRNAs were differentially expressed in cells exposed to thermal stress. We collectively refer to these miRNAs as thermalregulated microRNAs (TRMs). Since miRNA research is in its infancy, it is interesting to note that only 27 of the 123 TRMs are currently annotated in the Sanger miRNA registry. Prior to publication, we plan to submit the remaining novel 96 miRNA gene sequences for proper naming. Computational and thermodynamic modeling algorithms were employed to identify putative mRNA targets for the TRMs, and these studies predict that TRMs regulate the mRNA expression of various proteins that are involved in the cellular stress response. Future empirical studies will be conducted to validate these theoretical predictions, and to further examine the specific role that TRMs play in the cellular stress response.

Fluorescent microarrays have the ability to detect and monitor multiple analytes simultaneously and noninvasively, following initial placement. This versatility is advantageous for several biological applications including drug discovery, biohazard detection, transplant organ preservation and cell culture monitoring. In this work, poly(ethylene glycol) hydrogel microarrays are described that can be used to measure multiple analytes, including H+ and dissolved oxygen. The array elements are created by filling micro-channels with a hydrogel precursor solution containing analyte specific fluorescent sensors. A photomask is used to create the microarray through UV polymerization of the PEG precursor solution. A compact imaging system composed of a CCD camera, high powered LED, and two optical filters is used to measure the change in fluorescence emission corresponding to analyte concentration. The proposed system was tested in aqueous solution by altering relevant analyte concentrations across their biological ranges.

The overall goal for this project is the development and study of a quantitative fluorescence sensor for in vivo detection of β-amyloid (Aβ), the primary protein component of senile plaques in Alzheimer's disease (AD). Toward achieving that goal a Monte Carlo simulation has been developed to model photon propagation through the human head and a phantom model of the human head has been built and tested. In both cases a four layer model that included the skin, skull, fluorescent biosensor, and gray matter was used. A sensitivity study was performed to investigate the influence on the fluorescent output intensity of changes in concentration of the sensor. The results show that the fluorescent output intensity is detectable with a reasonable fluorescent sensor concentration and increases nearly linearly with increases in fluorescent concentration in the sensor. These results imply that the sensor would be detectable through the head using a reasonable optical system. The overall results are being used to aid in the design of the fluorescent sensor and the optical system for early detection of AD.

A minimally invasive biosensor is undergoing development to detect physiological concentrations of glucose within interstitial fluid. The sensor is based on a chemical assay consisting of Alexa Fluor 647 labeled concanavalin A lectin and dendrimer macromolecules functionalized to contain peripheral glucose moieties. The two components form large cross-linked particles that result in loss of fluorescent emission through shielding of interior fluorophores. As glucose is introduced into the assay, it competes with the glycodendrimers for binding to concanavalin A to disrupt the cross-linked complex and produce a reversible change in fluorescence intensity that is dependent on glucose concentration. Chemical analogs of the original glycodendrimer have been created and analyzed with the purpose of creating more stable and consistent dendrimers in order to maximize the response of the assay so that its signal can be better detected through dermal tissue and provide a better understanding of the sensor binding mechanics.

A combined dissolved oxygen and pH sensitive poly(ethylene glycol) hydrogel microarray was fabricated for use in monitoring cell culture media. The sensor was prepared by filling micro-channels with a hydrogel precursor solution containing pH or oxygen sensitive fluorophores. This solution was then cured by passing UV light through a mask placed to the channels, creating array with 100 μm elements. An imaging system with a monochrome CCD camera and appropriate interference filters was used to capture the fluorescence image induced by excitation of microstructure in transmission mode. The sensor performance was characterized in buffer solution (PBS) and cell culture media (MEM) across the biological range of pH (6-8) and dissolved oxygen (3-21%).

Poly(ethylene glycol) (PEG) microspheres have been used to sense a variety of analytes by encapsulating fluorescently labeled molecules into a PEG hydrogel matrix. This matrix is designed to retain the sensing molecules while simultaneously allowing nearly unhindered analyte diffusion. Some sensing assays, however, depend on the conformational rearrangement or binding of large macromolecular compounds which may be sterically prohibited in a dense polymer matrix. A new microporation process has been developed in order to create small cavities in the spheres containing aqueous solution and the assay components. This configuration insures a small mesh size for the supporting polymer, which limits leaching, while allowing the large assay components space to react within the aqueous cavities. Three hydrogel compositions (100% PEG, 50% PEG hydrogels, and microporated 100% PEG) were studied by embedding traditional pH (FITC) and oxygen sensitive fluorophores (Ru(Phen)). These hydrogels were analyzed for leaching and dynamic response to evaluate the functionality of the new microporated hydrogel.

A fluorescent assay based on the competitive binding between glycosylated PAMAM dendrimer and glucose with the sugar-binding lectin Concanavalin A has been developed. This assay, composed of the glycodendrimer and Alexa Fluor 647 labeled Concanavalin A, has shown a large dynamic response to physiological concentrations of glucose. The larger dynamic range is believed to be due to the spheriodal shape of the dendrimer molecule, which eliminates the multiple binding of the same dextran chain to the Concanavalin A tetramer that plagued previous approaches. However, in order to further understand the operation of the assay and optimize the dynamic response, the dendrimer construction must be modified to determine the optimum degree of glycosylation. In this paper, a description of the assay function and the change in fluorescence response with various formulations of glycodendrimers are shown. Theories are also presented as means of understanding the various assay responses with different degrees of dendrimer functionalization.

Fluorescent glucose assays based on the affinity reaction between Concanavalin A and dextran have been extensively studied. However, advancements in polymer science have allowed for new macromolecules capable of replacing dextran which may improve the performance of this well-known assay. Dendrimer macromolecules, being highly ordered and spherical, allow for the binding of specific residues to the terminal (peripheral) binding sites, enabling researchers to customize the molecule. In this research, glycosylated dendrimers have been engineered to replace dextran to allow for more controlled chemical and fluorescent responses (eliminate multivalent binding and improve reversibility). This new assay has been shown to form small aggregate particles containing many Con A and glycosylated dendrimers resulting in a substantial loss in fluorescent intensity. Overall, this assay shows promise for use as part of an implantable glucose monitoring device, but more research needs to be done to increase sensor stability and optimize the sensor response to glucose.

A blood perfusion and oxygenation sensor has been developed for in situ monitoring of transplanted organs. In processing in situ data, motion artifacts due to increased perfusion can create invalid oxygenation saturation values. In order to remove the unwanted artifacts from the pulsatile signal, adaptive filtering was employed using a third wavelength source centered at 810nm as a reference signal. The 810 nm source resides approximately at the isosbestic point in the hemoglobin absorption curve where the absorbance of light is nearly equal for oxygenated and deoxygenated hemoglobin. Using an autocorrelation based algorithm oxygenation saturation values can be obtained without the need for large sampling data sets allowing for near real-time processing. This technique has been shown to be more reliable than traditional techniques and proven to adequately improve the measurement of oxygenation values in varying perfusion states.

An implantable sensor is being created that allows measurement of blood glucose through fluorescent detection of an embedded chemical assay. The sensor is based on the competitive binding reaction between the protein Concanavalin A and various saccharide molecules, specifically a glycodendrimer and glucose. Previous studies have shown the ability of an embedded chemical assay using Con A and dextran with shorter wavelength dyes to both sense changes in glucose and generate sufficient fluorescent emission to pass through the dermal tissue. However, due to the chemical constituents of the assay, multivalent binding was evident resulting in poor spectral change due to glucose within the biological range. Use of a glycodendrimer and longer wavelength dyes has improved the sensor’s spectral change due to glucose and the overall signal to noise ratio of the sensor. In this work, a description of this sensor and the results obtained from it will be presented showing a large dynamic range of fluorescence with glucose.

A preliminary in vivo study using photopolymerized poly(ethylene glycol) (PEG) microspheres containing tetramethylrhodamine isothiocyanate labeld concanavalin A (TRITC-Con A) fluroescein isothiocyanate labeld dextran (FITX-dextran) as an implantable glucose sensor was performed using hairless rats. The glucose sensor works by affinity reaction between the two fluorescent labeled molecules binding together to form a fluorescent energy transfer system in which the FITC peak is quenched by the TRITC peak. The addition of glucose to the sensors local environment displces the dextran disrupting the FRET pair and the quenching. The change in fluroescent peak ratio (TRITC/FITC) therefore can be related to glucose. The microspheres in this study were implanted below the dermal skin layer of the lower abdomen by injection. A bolus injection of glucose was given through the tail vein to simulate glucose consumption. Spectra were obtained by shining and collecting light through the skin using an optical fiber delivery system via a 488nm argon laser and a spectrophometer. The preliminary results showed quantifiable changes in the ratio between the two peaks in response to the changae in glucose levels in the interstitial fluid of the rat.

A Monte Carlo method was developed to model light transport through multi-layered tissue with the application focused on the development of an implantable perfusion monitor. The model was developed and then verified experimentally with a micro perfusion phantom. The program modeled a three-layer (tissue, capillary bed, tissue) scenario to investigate the source-detector separation effects for an implantable sensor. The Monte Carlo code was used specifically to model the effects of absorption and scattering properties of the surrounding tissue, the hemoglobin concentration in the middle layer, the ratio of thickness of the capillary layer to the first layer, and the probe-source separation distance on the propagation of the light through the tissue. The model was verified experimentally, using a simple in vitro system with optical source and detector fibers separated at various distances. The model was also used to investigate fluctuations in luminance as a result of hemoglobin concentrations and the response of the system to various wavelengths. The model was helpful for an ongoing project to develop an implantable perfusion monitor for transplanted organs or skin flaps.© (2003) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Glucose monitoring is of critical importance in the life of Type I and many Type II diabetics. This research furthers work toward a minimally invasive implantable glucose sensor based on fluorescence detection. Current experimental models use heterogeneous fluorescence resonance energy transfer (FRET) systems for sensing; ideally, the response of one fluorophore bound to a large polysaccharide is enhanced greatly in the presence of glucose while the other fluorophore bound to a glucose sensitive protein is diminished or unaffected. Many fluorophores are affected by environmental factors such as pH and temperature. FRET experiments using two fluorophores, tetramethylrodamine isothiocyanate (TRITC) and fluoroscein isothiocyanate (FITC), are performed evaluating the effects of fluctuations over the range of pH 4-8 and temperature 25-45 degree(s)C for various concentrations of glucose in a flow cell. TRITC is bound to the lectin Concanavalin A (Con A), and FITC is bound to dextran molecules of varying sizes.