This PDF file contains the front matter associated with SPIE Proceedings Volume 11238 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Even though recent advances in medical devices have significantly improved clinical interventions, more accurate differentiation of biological tissues is still required to improve clinical decision-making. Tissue identification can be performed by using molecular-sensitive techniques such as diffuse reflectance spectroscopy (DRS), which is allows label-free, non-invasive, real-time and in situ interrogation of biological tissues. In this study, we used broadband DRS to extract biomolecule concentrations of gastrointestinal tissues and evaluated its potential for tissue identification and cancer detection. Diffuse reflectance spectra were analysed in an extended wavelength range between 350 nm and 1900 nm. This range covers the third optical window, which may allow better tissue identification for laparoscopy and gastrointestinal robotic surgery. Chromophore concentrations were obtained by using an inverse Monte Carlo Lookup table model to fit the reflectance spectrum.

Laser-induced plasma generation by single and multiple femtosecond laser pulses is used surgically and constitutes a source of photodamage in nonlinear microscopy. The irradiance threshold at which transient vapor bubbles in water are produced is 20x higher than the irradiance used for nonlinear microscopy. However, photodamage in multiphoton microscopy already starts, when the irradiance is 1.5x above autofluorescence imaging. Thus, there is a huge realm of low-density plasma effects between the multi-pulse damage threshold and the single-pulse surgical regime, and the talk will provide a systematic overview over laser applications and the irradiance and radiant exposure dependence of these effects.

Accurate sorting of specific particles in a mixed population is a desirable capability in the field of biomedical sciences. This enables researchers to purify samples by selecting only the particles of interest. Optical sorting is achieved by using a Bessel beam, which is a non-diffracting, propagation invariant light pattern consisting of concentric rings around a bright central core. This type of beam profile has the ability to employ optical forces in manipulating matter in a sterile environment without physical interaction. The concentric rings enable the simultaneous manipulation of particles of various characteristics in multiple planes due to the different power intensity distributions. Sorting with Bessel beam is an attractive approach using small sample volumes (microliter ranges), which becomes beneficial when working with rare particles of interest and in small samples. In this study a home built Bessel beam optical sorting setup was used to sort polystyrene and silica microspheres of different sizes and refractive indices. Our preliminary results showed that the polystyrene microspheres travelled quicker than the silica type of spheres with the same size due to the high refractive indices. These findings indicate the potential application of sorting different cells with varying refractive indices such as differentiating HIV infected cells from uninfected cells.

Medical applications of treatment, diagnosis and surgery can greatly benefit from the use of optical radiation. Every biomedical optical technique depends strongly on light propagation. The spatial configuration and the characteristics of optical radiation at each spatial point greatly influence the outcome of the previously mentioned applications. Light properties as it traverses biological tissues are particularly relevant in optical diagnosis. Diagnosis by optical radiation is usually based on pure intensity measurements. Consequently, there is a general lack of enough contrast, as it is based on pure absorption and scattering differences. Enhanced contrast can be achieved by taking into account other light parameters, such as coherence or polarization. These parameters present a much more complex evolution, and are strongly dependent on the incident optical beam properties, as long as on the biological medium characteristics. The statistical nature of the process makes it convenient to use random beams and even random media in the models. These additional parameters could represent the possibility to distinguish malignant from healthy biological tissues, when intensity contrast is not enough. What is more, beam characteristics could be chosen in order to produce desired spatial distributions of radiation inside biological tissues, or to provide an adequate interpretation of diagnostic parameters. In this work, optical random beams, mainly Gaussian-based, are employed to model light propagation in turbid biological tissues by Green’s functions. Coherence and spectral characteristics of the beam are considered. The model is applied to skin pathologies, such as basocellular or squamous cell carcinoma.

Optical systems emitting radiation in the visible and near infrared wavelength range represent a potential hazard for the retina of the human eye. This can result in irreparable damages due to photochemical, photothermal, photomechanical or thermomechanical interactions. To perform an eye safety evaluation a distinction must be made between coherent light sources and broadband light sources. For both types of light sources the corresponding safety standards, namely the IEC 60825-1:2014 and the IEC 62471:2006, provide accessible emission limits which depend on the angular subtense of the apparent source. This parameter is not a characteristic of the light source but must be determined by the irradiance distribution of the retinal image in case of coherent radiation and by the spectral radiance distribution of the retinal image in case of broadband radiation. This investigation introduces software-based methods investigating the retinal image in order to calculate the angular subtense of the apparent source. The results can be used to perform an eye safety evaluation in conformity with the laser safety standard IEC 60825-1:2014 as well as with the lamp safety standard IEC 62471:2006. However, the procedures given by the standards are not clearly defined. For this reason different implementations are discussed and compared to each other for a broad variety of exemplary retinal images.

Monte Carlo Simulations (MCSs) allow for the estimation of photon propagation through media given knowledge of the geometry and optical properties. Previous research has demonstrated that the inverse of this problem may be solved as well, where neural networks trained on photon distributions can be used to estimate refractive index, scattering and absorption coefficients. To extend this work, time-dependent MCSs are used to generate data sets of photon propagation through various media. These simulations were treated as stacks of 2D images in time and used to train convolutional networks to estimate tissue parameters. To find potential features that drive network performance on this task, networks were randomly generated. Generated networks were then trained. The networks were validated using 4-fold cross validation. The consistently performing top 10 networks typically had an emphasis on convolutional chains and convolutional chains ending in max pooling.

Systolic and diastolic blood pressure values can be used as an indicator of an individual’s risk for cardiovascular disease. The common practice of blood pressure (BP) measurement using a cuff-based system provides a snapshot of blood pressure at a single instance in time and can be inconvenient and intrusive. The development of optical methods to determine blood pressure could provide continuous monitoring of blood pressure through techniques such as pulse transit time (PTT) or pulse arrival time (PAT) when used with echocardiogram. Cuff based BP devices are known to have variation and inaccuracies when applied to larger arm sizes as seen in individuals with obesity but little is known of the influence of obesity in the PPG/PTT and PAT signals. We propose that accurate waveform replication is required for the derivation of blood pressure applied to individuals with obesity. Here we use the Monte Carlo framework to develop the PPG waveform as a means to derive blood pressure through cuff less techniques. The development of a simulated waveform incorporates realistic changes in the artery related to its biomechanical properties as a pressure wave is propagated through the vessel. It is shown that a change in vessel pressure and geometry directly affects the captured optical signal. The system can account for variations in body-mass index to compensate for geometrical changes in adipose tissue layer and changes in optical properties.

Tissue optics education can use several open-source tools available online. Even though these tools can be accessed by a broad student population, education environments, especially during outreach activities, may require more user-friendly and high speed tools that do not require previous programming experience. Then, quick estimators of tissue optical properties based on the tissue chromophore composition and analytical solvers based on diffusion equation can be a more suitable option for short events. In this study, we evaluated students experience during 1.5 hour of computer activities using a tissue-optics computer app. These activities were scheduled during a 7-hour biophotonics workshop given to undergraduate students in 2018 and 2019. Our results suggest the app is user-friendly and suitable for outreach activities. The use of our app may contribute to improve teaching and learning in biomedical optics and biophotonics.

Advances in fiber laser technology have resulted in the increased use and availability of several high-power laser systems operating in the mid-infrared band with short switching times and high modulation rates. The current American National Standard for the Safe Use of Lasers (ANSI Z136.1-2014) defines the calculation of the maximum permissible exposure (MPE) on the skin in terms of the exposure duration a single pulse or the total exposure time limit, and is based on continuous-wave laser skin exposure minimum visible lesion (MVL) data. This study determined the MVL data thresholds in Yucatan miniature pig skin for multiple-pulse 1940-nm laser exposures with pulse repetition frequencies (PRFs) of 100, 200, and 1000 Hz, and trains of 300, 1000, or 3000 pulses. The individual pulse duration in each exposure train was 500 µs. We report the MVL thresholds as the median effective dose (ED₅₀) based on varying individual pulse energy. The results highlight the effect of PRF on the thresholds for multiple-pulse cases. Comparison with the existing ANSI Z136.1 MPE limits provides a calculation of the safety margin for each parameter case.

Previous studies of laser-induced multiple-pulse damage measurements of the retina determined inconsistent thresholds in the thermo-mechanical damage regime. This inconsistency might be due to biological variability, measurement techniques as well as methodological challenges which occur during the use of explants. For better comparability with state-of-the-art measurements, we use a top hat irradiance profile to produce a uniform energy density and monitor the beam profile at the retinal pigment epithelium layer. This approach is applied in order to determine damage thresholds in the lower nanosecond regime for pulse sequences. The irradiation experiments were performed using a Q-switched, frequency-doubled Nd:YAG laser (532 nm wavelength, 1.8 ns pulse, 25 Hz, 319 μm-diameter, squared top hat). Freshly isolated porcine eyes were used as models for measurements of laserinduced thermo-mechanical damage. After removal of the sensory retina the retinal pigment epithelium layer attached to the sclera was irradiated. Four pulse trains (N = 1, 10, 100 and 1 000) were examined and evaluated concerning damage threshold via fluorescence microscopy. The ED₅₀, expressed as energy per pulse, decreases from 33.9 J for N = 1 to 20.3 J for N = 1 000. The multiple-pulse threshold trend which was measured can neither be described by current methods such as the probability-summation-model nor by the N−^0,25- approach which is also partly used within the laser safety standard. Therefore we discuss possible pulse interactions which could explain the multiple-pulse behavior in the thermo-mechanical damage regime. Their implications might present the build-up of a new, more accurate theory to describe the reduction factors of thermo-mechanical damage-inducing doses with increasing pulse numbers.

Multiple commercial femtosecond lasers have been cleared for use by the US Food and Drug Administration for ophthalmic surgery, including use in creating corneal flaps in LASIK surgery. The newest application of femtosecond lasers in ophthalmology is in cataract surgery. Currently there are a few lasers at or near the point of commercial release. LenSx (Alcon Laboratories Inc., Ft Worth, TX, USA) is the first one which get FDA permit and most popular one in the clinic. During normal operation, some of laser energy passes beyond the cornea and through the lens with potential effects on the retina. As a model for retinal laser exposure during OCT assisted femtosecond laser surgery, we measured the temperature rise in human cadaver retinas during direct illumination by the laser. Human cadaver retinas were irradiated with a LenSx femtosecond laser and the temperature rise was measured with an infrared thermal camera. The results showed a temperature rise of less than 0.5 degrees for realistic pulse energies. A numerical simulation was developed to quantify the temperature rise as a validation of the ex-vivo experiments. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser cataract surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage.

Achieving functionalized artificial skin with immune response would solve an important problem when treating burn patients with larger skin transplants, since the skin is the main immune barrier of the human body and until now these models have lacked immune response. Furthermore, with the proper biological approach, we could obtain a generic skin suitable for transplantation to patients with different major histocompatibility complex (MHC). This work presents an original method to achieve this goal by creating cylindrical pore-like structures with laser, where subsequently immune cells are included using a BA-LIFT (Blister Actuated Laser Induced Forward Transfer) technique in a co-culture with the treated skin, in order to assess their engraftment to the artificial skin.

The removal of tissue with a laser scalpel is a complex process that is affected by the laser incidence angle on the surface of the tissue. Current models of laser ablation, however, do not account for the angle of incidence, assuming that it is always normal to the surface. In order to improve ablation modeling in soft tissue, this work characterizes photoablation crater profiles at incidence angles ranging from 0 degrees to 45 degrees off perpendicular. Simulated results, based on a discretized steady-state ablation model, are generated for comparison based on the assumption that material removal occurs in the direction of the laser. Experiments in an agarose-based, homogeneous soft tissue phantom are performed with a carbon dioxide (CO₂) laser. Surface profiles of the craters are acquired using a micro x-ray computed tomography scanner (Micro-CT) and compared to results from the simulation. The difference of the simulated and experimental results are measured and the error analysis is reported.

Laser ablation (LA) has shown promising results in selective treatment of solid tumors. Recently, nanomaterials, in particular nanoparticles (NPs), have been proposed as mediators for laser tissue ablation due to their high optical absorption coefficients. In this work, we report distributed fiber optic temperature sensors for monitoring of NPmediated LA in ex-vivo porcine liver. This study aims at improving the outcomes of LA through magnetite NPsenhanced LA with in-situ thermal profiling. Such thermal profiling is achieved with optical backscatter reflectometer interrogating a set of custom-made MgO-doped optical fibers exhibiting enhanced scattering profiles. Fiber optic sensors, providing spatially resolved measurements, significantly outperform conventional thermocouples and imaging techniques. A minimally invasive LA setup is based on high power fiber coupled diode laser operating at 980 nm wavelength, with output power up to 30 W. Magnetite (Fe₃O₄) NPs are synthetized and locally injected within the tissue before performing LA. The sensing setup utilizes optical backscatter reflectometer that exploits Rayleigh backscattering to measure the temperature distribution with submillimeter spacing. Thermal maps, i.e. temperature distribution as a function of space and time, are reported highlighting thermal distribution within the ablated lesion and in off-target adjacent tissue. The influence of laser power and of NPs concentrations on the outcomes of LA is also investigated. Results demonstrate that injection of NPs into targeted area helps enhance conversion of light energy into thermal energy, thus increasing the efficacy of the ablation within the treated area, without overheating the adjacent off-target tissue.

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 confocal microscope system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 10-50 kV/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.

We obtain the pathlength distribution of detected photons in single fiber reflectance spectroscopy (SFR) from reflectance as a function of absorption coefficient using inverse Laplace transformation. We recently developed a novel model, writing the SFR signal in terms of a semi-ballistic and diffuse component. Here, we study the pathlength distributions of both components; and compare those to the path length distribution corresponding to a previously available empirical model. We will explore possible scaling relationships that follow from the Laplace relation for reflectance and the pathlength distribution in terms of the absorption coefficient, reduced scattering coefficient and fiber diameter.

Raman imaging continues to grow in popularity as a label-free technique for characterizing the underlying chemical structure of biological materials, both in-vitro and in-vivo. While Raman spectra demonstrate high chemical specificity, spontaneous Raman scattering is an inherently weak process and requires prohibitively long acquisition times. When Raman is utilized to image highly scattering cellular environments, integration times can be on the order of several minutes to hours. Recently developed compressed sensing techniques can greatly improve hyperspectral Raman acquisition times by randomly under-sampling the spatial dimensions. A digital micromirror device (DMD) is used to spatially encode the image plane. The encoded image is then propagated to a spectrometer where the spectral components are produced by shearing one spatial dimension. Several reconstruction algorithms have been developed that can then be used to return the original. Here, we will present single-shot, 2D Raman imaging of CHO cells using compressed hyperspectral Raman microscope. This system provides an order of magnitude improvement on traditional hyperspectral acquisition rates. Single-shot compressed hyperspectral Raman images can reveal biochemical changes due to short lifetime dynamic processes. These improvements will allow imaging of samples that metabolize quickly, rapidly oxidize, or are physically altered under experimental conditions.

Mueller matrix polar decomposition (MMPD) is capable of extracting intrinsic polarimetric characteristics of tissue-like media, including the orientation angle of the linear retardance axis. Here we propose a modified MMPD parameter to calculate linear birefringence orientation for multi-layered media. By measuring different experimental layered phantoms designed by ourselves and pathological tissue slices, we demonstrate that the calibrated new parameter can show more accurately the fast axis orientation angle. Since the multi-layered anisotropic structures are widely distributed in biomedical samples, we believe that the parameter proposed in this study can be helpful for future biomedical diagnosis.

We present an extension to existing Monte Carlo photon transport methods to simulate integrating sphere experiments. This method uses a Monte Carlo approach to simulate photon paths in tissue and an analytical expression for the probability of a photon in an integrating sphere being re-incident on the tissue. Analytical models, previous works on Monte Carlo photon transport, and measurements of a synthetic tissue phantom validate this method. We present two approaches to back-calculate the optical properties of samples. Experimental and simulation uncertainties are propagated through both methods. Both back-calculation methods find the optical properties of a sample accurately and precisely.

Here, we present a technique for inverting a Monte Carlo simulation to extract tissue optical properties from the moments of the spatio-temporal response of the tissue by training a 5-layer fully connected neural network. Initial reports of this model were used to extract optical properties from a single layer tissue model with high accuracy. Here, we expand this method to demonstrate its ability to extract optical properties from individual layers in a multi-layer model. We demonstrate the accuracy of the method across a very wide parameter space and demonstrate that the method is insensitive to parameter selection of the neural network model itself.

With the highest mortality rate among rheumatic diseases, scleroderma leads to the painful formation of thick skin and organs. The cause of this thickening is the overproduction of collagen protein in the skin. The pathogenesis scleroderma is not fully understood and there exist no good disese models to provide context for drug discovery. To tackle this problem, we created a disease model of scleroderma with 3D printed collagen containing fibroblasts, MATLAB and confocal microscopy to observe optical properties mu and rho, which can be directly mapped to the scattering coefficient and scattering anisotropy. These optical properties varied with collagen levels and served as our metric for disease model severity. Our observations were based on the correlation between Rho and Mu values in the decay -- Signal = Rho*e(-Mu*Z) -- of the light absorbed over the depth of the tissue. We found that in our scleroderma model the Rho and Mu values increased with age and collagen concentration.

Since regular biological tissue’s optical characteristics are unstable building long lived test phantoms that simulate their scattering characteristics are needed for testing optical methods of imaging through tissue. Our ongoing work developed a methodology to build long term stable phantoms with lifetimes >6 years which can be tuned to maintaining optical characteristics mimicing skin characteristics and a rapid technique to measure the scattering coefficients μ_s and anisotropy factor g. Our test phantoms employ intralipid-infused agar layers 1 to 8 mm thick. Agra can take a wide range of intralipid concentrations enabling the building phantoms of a large range of scattering parameters with typical values of μ_s=20cm^-1, g=0.95. Encapsulating the intralipid-infused agar within a clear polymer has proved to stabilize these for long lifetimes and allows creation of varying thicknesses, scattering characteristics and shapes. We developed a rapid technique where the light from a laser beam passing perpendicular through the test phantom is captured using a 36×24mm digital camera sensor. This gathers ~6×10⁶ measurements over a ±12° range, giving ~20,000 points each at 2300 angular bins of 0.005° . A Matlab program identifies the scattering center and data points for each angular position. Using a HenyeyGreenstein two-term model a nonlinear curve fitting extracts pairs of HG weighing factors, µs and g parameters. Fit results show extremely high statistical significance with exceedingly small deviation from the HG model for multiple wavelengths (currently 533, 632, 670 nm lasers and a supercontinuum laser at 650, 700,750 nm) for several test phantoms.

Reflectance spectroscopy shows itself as a useful tool to characterize turbid media, such as biological tissues. The light backscattered from the medium is usually collected by imaging systems or optical fiber probes. In this work we used an optical fiber probe, with a linear arrangement of the source and detection fibers that allows spatially resolved reflectance (SRR) measurements. Through the use of inverse model, the collected SRR can be exploited to estimate the optical properties of the turbid medium. The estimation process involves matching of the measured and simulated SRR that accounts for all the details of the measurement setting. At small source-detector separations and/or non-negligible absorbance, the reflectance becomes highly dependent on the scattering phase function of the medium, which can be efficiently described by the higher order Legendre moments and related scattering phase function quantifiers (PFQ). In our previous studies, we utilized the Gegenbauer Kernel (GK) scattering phase function to describe the light propagation in turbid samples. However, the domain of GK-based PFQs is quite small and fails to fully encompass the scattering phase functions of microspherical suspensions, typically used for calibration and validation of SRR measurement settings. This limitation could be overcome by utilizing scattering phase function models with a large PFQ domain that may also lead to more accurate and robust inverse model predictions. To verify this hypothesis, we evaluate various scattering phase function models that maximize the PFQ domain and experimentally validate the inverse models by SRR collected from optical phantoms and various turbid samples.

Neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers are among the most commonly used lasers with a wide variety of applications from biomedicine to manufacturing. The ubiquity of these lasers increases the likelihood of accidental ocular injury resulting in permanent visual impairment. We performed dosimetry studies to determine retinal damage thresholds and hemorrhagic lesions in the porcine eye with Qswitched Nd:YAG lasers. The Yucatan miniature pig model exhibited similarities in ocular anatomy to human eyes. The Nd:YAG laser, tuned to 1064 nm with a pulse width of seven nanoseconds, delivered laser energy to the retina. Retinal imaging modalities including fundus photography, real-time video, confocal scanning laser ophthalmoscopy (cSLO), and spectral domain optical coherence tomography (SD-OCT) provided visualization of retinal morphology at multiple time points. Retinal damage thresholds were grouped into three categories: minimum visible lesion (MVL), contained hemorrhagic lesion (CHL), and vitreal hemorrhagic lesion (VHL). Probit analysis determined the effective dose for 50% probability of damage (ED₅₀) for each lesion category. The threshold to produce a MVL was 0.193 mJ based on 24-hour assessments of the retina. The one-hour hemorrhagic lesion thresholds were 0.408 mJ and 1.52 mJ for CHL and VHL, respectively

Multiple commercial femtosecond lasers have been cleared for use by the US Food and Drug Administration for ophthalmic surgery, including use in creating corneal flaps in LASIK surgery. The newest application of femtosecond lasers in ophthalmology is in cataract surgery. Currently there are a few lasers at or near the point of commercial release. LenSx (Alcon Laboratories Inc., Ft Worth, TX, USA) is the first one which get FDA permit and most popular one in the clinic. During normal operation, some of laser energy passes beyond the cornea and through the lens with potential effects on the retina. As a model for retinal laser exposure during OCT assisted femtosecond laser surgery, we measured the temperature rise in human cadaver retinas during direct illumination by the laser. Human cadaver retinas were irradiated with a LenSx femtosecond laser and the temperature rise was measured with an infrared thermal camera. The results showed a temperature rise of less than 0.5 degrees for realistic pulse energies. A numerical simulation was developed to quantify the temperature rise as a validation of the ex-vivo experiments. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser cataract surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage.

Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, a nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, a nsPEF exposure can activate receptors and channels on the membrane as well as second messenger cascades, both of which results in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the extent/sequence of physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 mouse myoblast cells have been demonstrated to be a useful in vitro model, as it is feasible to differentiate these immortalized progenitors into terminally transformed myotubes. From previous efforts, we quantified YO-PRO -1 (YO-PRO-1) uptake as a measurement of membrane perturbation, and concluded that membrane damage is proportional to applied pulsed electric field voltage. To expand upon these findings, we evaluated to what extent YOPRO-1 uptake at the membrane is physical or physiological in nature. Interestingly, the P2X7 receptor complex has been extensively studied utilizing YO-PRO-1 uptake as marker of apoptotic activity. For this reason, we tested the role of P2X7 receptor complex activation to mediate YO-PRO-1 uptake during pulsed electric field exposure. By blocking the P2X7 receptor, we reduced nsPEF-induced YO-PRO-1 uptake by 31.57%. Our results demonstrate that the P2X7 receptor complex is a subcellular candidate responsible for YO-PRO-1 uptake upon nsPEF exposure in myotubes.

Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, nsPEF exposure can activate second messenger cascades resulting in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 immortalized mouse myoblast cells have been demonstrated to be a useful in vitro model, by differentiating these progenitors into terminally transformed myotubes. We are not only able to further investigate the fundamental subcellular mechanisms activated by pulsed electric fields, but monitor muscle contraction as a functional output. From our previous efforts, we quantified calcium-green uptake as a measurement of cellular calcium uptake across a sweep of applied pulsed electric field voltages. To extend on these findings, we evaluated calcium dynamics in the intracellular space of myotubes. Given that sarcoplasmic reticulum efflux is required for muscle contraction, we tested the physiological role of the ryanodine receptor during pulsed electric field exposure on myotubes. By blocking the Ryanodine receptor with a competitive antagonist, we reduced nsPEF -induced calcium dynamics activation by 58.36% in media with calcium. Our results are the first to demonstrate that the Ryanodine receptor complex is a subcellular candidate responsible for generating calcium responses upon nsPEF exposure in myotubes.

Infrared laser (IRL) exposure can induce a rapid temperature change (fast thermal gradient or FTG) that is able to stimulate or inhibit neurons and, thereby, modify neurological functions. Despite extensive research into this effect, the fundamental mechanism(s) underlying how FTG causes neurological stimulation or inhibition remains unclear. While it is hypothesized that IRL-induced FTG acts directly on the neuronal plasma membrane (PM), it is uncertain if the neurological effects observed in previous studies are mostly derived from presynaptic effects (i.e., modifications in action potential (AP) firing) or also from postsynaptic effects (i.e., alteration of the synaptic responses of the excitatory and inhibitory neuronal receptors). In the present study, we present an analysis of FTG-mediated changes in neuronal PM, AP firing rate, and miniature postsynaptic excitatory and inhibitory currents (mEPSCs and mIPSCs). Our results suggest FTG induces changes in both presynaptic and postsynaptic neurophysiological mechanisms. Specifically, we found that, after IRL pulse (IRLP)-induced FTG exposure, the amplitudes of APs are reduced, but the rate of APs are increased. In contrast, the quantities of both mEPSCs and mIPSCs are reduced, but the peak-to-peak frequency and peak amplitudes are increased. The results outlined in this study demonstrate the impact of FTG on neurons and neuronal network. This information is critical for understanding the complexity of the effects of FTG on neurological functions and for demonstrating how post-synaptic mechanisms might play a crucial role in neurological excitation or inhibition seen following IRL pulse exposure.

An infrared laser pulse (IRLP) can trigger the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), presumably via initial activation of phospholipase C (PLC), and activate multiple intracellular signaling cascades. Two main hydrolysis products are important second messengers, lipid diacylglycerol (DAG), that can lead to PKC activation, and soluble inositol 1,4,5-triphosphate (IP3) that stimulates calcium release from intracellular stores. The mobilization of these messengers can modulate ion conductivity through channels, and coordinate cytoskeletal rearrangements that promotes or suppresses further downstream signaling. The downstream effect impacts membrane conductance, membrane potential and overall cellular excitability. As a result, a single IRLP with a 2-4 millisecond duration may initiate a cellular effect that lasts for seconds to minutes. From our approach, we evaluated the PIP₂ phosphoinositide signaling cascade from immortalized cells that exhibited genetically encoded reporters for PIP₂/IP₃ and DAG. Upon IRLP exposure, we observed a PIP₂ depletion, IP₃ increase and DAG decrease in cytosol in motor neuron-like NG108 cells. Our data suggest that IRLP may induce second messenger systems at the membrane, and as a result modulate ionic signaling across the cell body.

We have assembled a double-integrating-sphere system to measure the absorption (μ_a) and reduced scattering (μ'_s) coefficients of materials with high turbidity. We have employed the Inverse Adding-Doubling (IAD) algorithm to access these optical properties. The experimental system is a homemade setup assembled with broadband sources and 3D printed spheres, equipped with an optical fiber bundle for excitation and a spectrometer for detection. We have used a reference phantom (polyurethane plus Titanium Dioxide) with known optical properties at 633 nm (μ_a= 0.0019 mm^-1 and μ'_s= 0.477 mm^-1 ) to validate the system. The system can reliably measure the optical properties of turbid samples from 400 to 950 nm with one of the experimental setups and from 950 to 1650 nm with a second experimental setup.

The accuracy of non-invasive detection devices using photoplethysmography signal (PPG) for blood content often fails to meet the medical clinical standards. The reason for the error is partly due to theoretical algorithms, and partly due to the design of hardware. PPG signal acquisition device puts pressure on the skin during measurement, which affects the PPG signal. Aiming at this problem, this paper uses the finite element method to construct a skin model under pressure, and the optical transmission simulation experiment are used to analyze the changing trend of the reflected light intensity under different pressures. It was found that the change of reflected light intensity with pressure is related to the detection distance and wavelength. Simultaneously, the PPG sensor in our laboratory are used to carry out pressure experiments. The measured results verify simulation results. The influence of pressure on the DC, AC component and quality of PPG signals are analyzed ulteriorly. And we found the optimal pressure range is 0.4N~1.2N for 7 subjects.

In recent years, it has been found that sound also has effect on plant growth and its yield with certain sound directing the seedling of corn toward the sound source and its ability in distinguishing stuttering of larvae from other sounds. However, methods investigating the effects of sound either take a long time or destructive. Here, we have used laser biospeckle, a non-destructive and non-contact technique to investigate the activities of an arugula plant (2-4 weeks old) under sounds of different frequencies of 0 Hz or control, 100 Hz, 1 kHz, 10 kHz. Laser biospeckle has been proved to be valid for investigating the aging of fruits, believed to be due to the movement of water, organelles etc.. Biospeckle activities were recorded for 20 sec at 15 fps following exposure to sounds for 1min. The correlation parameter (r) of biospeckle activity was used to characterize the activity of the plant with r being 1 for highly active and 0 for reduced plant activity. Sound level of 100 dB was the same for all frequencies. There was a clear difference in r between the control and other frequencies and r was lower than that of control indicating a reduction in the activity. Moreover, r for 100 Hz was found to be closer to control while at higher frequencies, r was much lower indicating a dependence of the activity on the frequency.

Electrical nerve stimulation (ENS) technique has been tested on nerve mapping devices, which are intraoperative diagnostic tools. However, these technologies suffer from general limitations. Optical Nerve Stimulation (ONS) has been a developing technique as a potential alternative to ENS. This new technique using infrared laser radiation can offer many advantages, including a non-contact stimulation mode, improved spatial selectivity, and elimination of stimulation artifacts. However, the stimulation parameters, including laser power, beam diameter, and surface scanning speed, provide a large variable matrix that must be optimized for consistent and reliable nerve mapping using ONS. This preliminary study explores a computational tool to provide a guiding map for determining optimal stimulation parameters for laser-scanning subsurface ONS. It consisted of three parts: (1) Monte Carlo simulations for generating laser energy distribution in the tissue sample, (2) laser-scanning model by moving the heat source at the surface, and (3) thermal transfer simulations to calculate the tissue temperature. The tool was tested on laser wavelengths of 1455 nm, 1490 nm, and 1550 nm. According to the results of calculations, nerve temperature maps were generated for those wavelengths. Each map demonstrated specific optimal parameter values to reach the nerve activation temperature. Additionally, the results of laser power densities at the lowest scanning speeds of 0.4 mm/s in x-axis and 0.5 mm/s in yaxis showed proximate results with our previous study about ONS on rat model. With further development, this tool may hold promise in the development of an intraoperative optical stimulus device for surgical applications.

Statistical interferometer technique (SIT) is a highly sensitive optical interferometer developed by us capable of measuring sub-nanometer displacements and when applied to plant growth studies revealed nanometric intrinsic fluctuations (NIF). NIF observed in minimum time scale of several tens of seconds is strongly influenced by the environmental conditions. Our earlier experiments with rice under ozone or heavy metal stress, such as cadmium even for a short duration of one hour decreased NIF. In contrast, having a micronutrient, such as zinc increased NIF. Therefore, presence of NIF is found to be a novel phenomenon characterizing plant condition that could appear only under sub-nanometric measurement. In this study, we report the effects of adding a plant growth hormone called auxin. Roots of rice seedlings were exposed to auxin solutions of different concentrations of 0, 1, and 4 μM for 24 hours. Significant increment was seen in NIF for a concentration of 1 μM while a significant reduction was seen in NIF for 4 μM within an hour after immersion of the roots. Application of an inhibitor to auxin called TIBA also resulted in almost immediate reduction of NIF. Current results suggest for NIF affected by the enodgenous hormones to be related to growth, as the action of a growth-related endogenous hormone auxin is chemically inactivated. Thus, NIF not only could be applied to investigate and speedily assess the effects of environmental agents on plant elongation or shrinkage but also could be implicated as one of the possible mechanisms for the origin of NIF itself.

The vagus nerve originating from the brainstem in the central nervous system is a long cranial nerve that reaches the neck, thorax, abdomen, and colon. It plays a role in autonomic nervous, cardiovascular, gastrointestinal, and immune systems. Electrical stimulation of the vagus nerve has become a standard method for the treatment of neuropathic pain and epileptic conditions over the years. Infrared laser nerve stimulation (ILNS) is an evolving technique that uses infrared laser energy to stimulate cells with electrochemical capacity without the need for external agents or physical contact. This pilot study explores infrared laser stimulation of the rat vagus nerve, in-vivo. An infrared pigtailed singlemode diode laser operating at 1505 nm in continuous-wave (CW) mode was used in this study for noncontact CW-ILNS. Successful CW-ILNS of the rat vagus nerve was observed after the CN reached a threshold temperature of ~44°C with response times as short as 10 s. With more improvement in instrumentation, better optimization of stimulation parameters, and a higher sample size, CW-ILNS may show some potential in vagus nerve stimulation for preclinical.

Laser vaporization is a surgical procedure which utilizes a high power laser to quickly heat and vaporize tissue. Laser vaporization can be conducted on internal organs, such as breast or prostate, through a fiber catheter. Compared with other surgical technologies, it has excellent hemostasis capability with minimal collateral tissue damage, which may reduce hospitalization time and postoperative complications. Accurately monitoring tissue temperature during laser vaporization procedures provides important feedback to surgeons to improve surgical outcomes. Tissue cannot be vaporized if the temperature is lower than the boiling point, while high temperatures may lead to carbonization over the tissue surface, which not only reduces vaporization efficiency but also leads to postsurgical complications. However, until now, no sensing technologies have been developed to monitor tissue temperature during routine laser vaporization in clinics. Here, we report the use of blackbody radiation in the short-wave infrared range (SWIR) for tissue temperature monitoring during laser vaporization. Although blackbody radiation in SWIR is very weak for temperatures less than 100°C, the relatively low water absorption and silica fiber attenuation may allow temperature sensing in vivo. We successfully detected blackbody radiation in SWIR down to 80°C through a 2 m silica fiber. We then proved the feasibility of using blackbody radiation in SWIR to monitor tissue temperature during laser vaporization through an ex vivo tissue study. The developed technology is low-cost and can be seamlessly integrated with the fiber catheter used in laser vaporization.

Current standards for diagnosing and monitoring anemia are relatively invasive. The superficial symptoms of this condition are due to an underlying deficiency of red blood cells (RBC) or erythrocytes, and hemoglobin in the blood. This results in an inadequate supply of oxygen to the body’s tissues. For point-of-care diagnostic systems, remote determination of blood conditions will depend on an understanding of the interaction of light with hemoglobin. However, the skin acts as the first barrier for this detection. In this study, we pursue the possibility of detecting anemic conditions from the perfused blood in the dermis using optical models and Monte Carlo (MC) methods. The skin is composed of two primary layers, the epidermis and the dermis. The avascular epidermis absorbs light due to its primary chromophore, melanin. Subsequently, the absorption in the dermis layer is quantified by hematocrit and hemoglobin concentrations. Two-layer models of the human skin are set up and optical properties are assigned to these models. The optical variability across these models are defined by six melanin (epidermis) and two erythrocytes (dermis) concentrations. The twelve combinations of optical properties are assessed at six wavelengths of interest in the Virtual Tissue Simulator (VTS) environment. The chosen wavelengths range across the visible and near-infrared spectrum, which is a known and important diagnostic window for biological tissues. In this study, we explore the variability of light interactions for healthy and anemic blood conditions quantified in the dermis while accounting for variable melanin concentrations in the epidermis.

In recent years, laser radiation has been proposed as a therapy tool in the medical field (laser therapy). This work presents the efficiency of a pulsed fiber optic laser in its interaction with synthetic monosodium urate monohydrate (MSUM) crystals, for the management of inflammatory processes and lesions in tissues, muscles and osteoarticular joints affected by crystalline pathologies. The synthetic monosodium urate crystals were made based on the McCarthy method. Such samples were exposed to the direct radiation of the laser light source, whose characteristics are as follows: wavelength centered at λ = 1058 nm, pulse width of τ = 13 ns, repetition frequency of f_R = 210 kHz and peak energy of E_P = 55 nJ. This radiation is absorbed by the tissue, which produces a photochemical interaction at the molecular level in the crystalline formations. The exposure times were from 7 to 12 minutes, and the best exposure time was presented in 12 m. As a result, the photochemical interaction of light with crystals could be observed, resulting in a variation in molecular structure. Likewise, a decrease in their concentration was found in order to make uric acid crystals, that are within the joints or tissues affected by crystalline pathologies, more soluble to be eliminated through the urine, and therefore, provide at the same time analgesic and anti-inflammatory action in the affected area.

Recent studies suggest that microtubules (MTs) and tubulin proteins exhibit resonant frequencies in the radiofrequency (RF) range. We hypothesize that exposing neurons to externally applied RF waves tuned to an intrinsic resonant frequency of MTs or tubulin could disrupt the natural signaling occurring in and around them, leading to neurophysiological changes. To test this hypothesis, we assembled custom exposure systems that allow stable RF exposures of cell cultures in a controlled environment (37°C, 5% CO₂, 95% humidity). We then exposed differentiated NG108-15 neuronal cells to RF waves tuned to selected resonance peaks for tubulin (91 MHz and 281 MHz) and for MTs (3.0 GHz) for 1 hr at a power density of 0.24 mW/cm² (SAR = 0.012, 0.087, and 0.53 mW/kg, respectively). We used fluorescence imaging of endogenous MTs and current-clamp electrophysiology to investigate changes following RF exposures compared to sham. The results from the imaging data show a clear difference in the localization of fluorescent MTs between the sham and the RF exposed neuronal cells. The sham cells exhibited more fluorescence in the neurite projections, whereas the RF exposed cells showed a more diffuse pattern, with a stronger fluorescence in the cell body. The electrophysiological results showed that resting membrane potentials of the RF exposed neuronal cells were more depolarized than those of the sham cells. Consequently, we observed spontaneous action potentials in the RF exposed cells, which were not present in the sham cells. Overall, our results suggest that exposing neurons to MTs or tubulin resonant frequencies might affect MTs normal behavior, leading to neurophysiological changes. However, to confirm the specificity of resonant frequency effect and validate this idea, studies investigating exposures to nonresonant frequencies and additional tubulin and MTs resonant frequencies are warranted.

Curcumin is a natural compound used for medical and food-industrial applications due to its antioxidant, antiinflammatory, antifungal, antiviral, and antiseptic properties. In addition to these applications, curcumin can be used for fluorescence labeling in disease diagnostics/theranostics and monitoring photodynamic antimicrobial inactivation. In terms of fluorescence-based applications, the sensitivity to detect curcumin depends on the curcumin bioavailability or the formulation concentration. In this study, we used 375 nm and 445 nm laser excitation to characterize the wavelength dependent fluorescence spectra for curcumin in ethanol solutions of 10, 20, 50 and 75 μg/ml (i.e., 27.15, 54.30, 135.75 and 203.625 μM, respectively). Our results suggested the fluorescence intensity as a function of concentration saturates around 135.75 μM. Fluorescence intensity increase as a function of concentration was observed between 650 nm and 800 nm for the 445 nm excitation. Increase in the same wavelength range was obtained for the difference between the fluorescence spectra generated between the 375 nm and 445 nm excitation wavelengths. The spectral features reported in our study can be used on the design of curcumin formulations using Sigma Aldrich curcumin C7727 for fluorescencebased applications, determination of bioavailability of curcumin formulations and photodynamic dosimetry.

The assessment of tooth color is typically performed by subjective comparison with a visual shade guide or by using objective optical techniques such as quantitative light-induced fluorescence (QLF). QLF measurements rely on the precise wavelength calibration of fluorescence excitation and emission for enhancing the contrast between the white sound tooth and stained areas. These areas may change the fluorescence emission differently depending on the color that is most absorbed by the stain on the tooth surface. Although previous studies have monitored the staining contrast generated by the consumption of beverages on teeth, the information provided is based on total intensity. However, this intensity varies from each QLF device configuration and comparison across studies may not be possible. Few studies report the wavelength-dependent characterization of the staining process, which allow the comparison on the light attenuation on specific wavelengths and can be used to design fluorescence equipment with improved contrast for the tooth color assessment. In this study, we quantified the fluorescence spectral features (fluorescence intensity, wavelength shift of the maximum intensity, full width at half maximum, and wavelength-dependent intensity attenuation) of teeth in several degrees of coffee pigmentation by using 445 nm excitation. Most of the pigmentation effect was observed on the fluorescence intensity and a linear behavior was observed for the full width at half maximum (around 11.8% increase for each pigmentation level). We characterized the fluorescence properties of each degree of pigmentation level. Both spectral features and fluorescence properties can be used to design novel fluorescence equipment capable of increasing the contrast between white and stained teeth.

Accurate color assessment is still a challenge for food-industrial applications and dental aesthetics. Even though current tooth color determination relies mostly on visual shade matching with shade guides, the technological advancements move towards objective tooth color assessment by using instruments such as colorimeters, spectrophotometers and digital cameras. Objective assessment of tooth color improves the communication between the professionals including clinicians, dentists, laboratory technicians and equipment designers. Tooth color can be evaluated by calculation of whiteness and yellowness indexes. Precise determination of these indexes is particularly important in dentistry, where monitoring the quality of dental restoration and the tooth appearance during whitening treatments is essential for improving the patient outcome. In this study, we evaluated the effect of tooth staining and a whitening treatment using violet illumination alone on yellowness and whiteness indexes. These indexes were quantified by colorimetry and digital photography, which were compared in terms of absolute values of the indexes and variation due to staining and whitening procedures. The violet illumination was capable of generating (36 ± 2) % of W* whiteness index recovery and (41 ± 2) % decrease on the YIE313 yellowness index. Even though the absolute W* and YIE313 values are relatively close for photographic and colorimetric methods, the indexes contrast were (37.3 ± 0.01) % and (12.8 ± 0.1) % lower for digital photography compared to colorimetry. We believe our study can be used as a guide for the evaluation of the color contrast generated on whitening monitoring devices in future studies.

We investigated light attenuation in a salted cadaver brain at 664 nm, which is the excitation wavelength of photodynamic therapy using Talaporfin sodium. Technology for diagnosis or treatment using light such as photodiagnosis and photodynamic therapy has been spread recently. Especially the therapeutic lesion by photodynamic therapy is dominated by the light distribution in the tissue under the case of uniform photosensitizer distribution. Estimation of therapeutic lesions is important to ensure the effectiveness and safety of brain tumor photodynamic therapy since important parts that should not be damaged are adjacent. Previously reported optical properties of the human brain in the literature vary widely, since the preparation and measurement methods are different. In this study, we measured the light attenuation in a salted cadaver brain using a practical method. In the employed salted cadaver brain, the mechanical and optical properties could be maintained as close as possible to those under operative conditions in living patients. Until the cerebral ventricle was reached, a neuroendoscope was inserted into the brain. A thin diffuse irradiation probe of 10 mm in irradiation length was inserted using the endoscope and advanced 10 mm from the endoscope tip. An optical fiber for measurement was inserted by another path from the brain surface. Optical fiber was put into a puncture needle, and a pair of needles was used to puncture the tissue and reach the same cerebral ventricle in which the endoscope tip and diffuse irradiation probe were positioned. Attenuation at 664 nm in salted cadaver brain in situ was reasonably measured by the withdrawal technique and we think this method might be appropriate for the measurement of inhomogeneous biological tissues.