This PDF file contains the front matter associated with SPIE Proceedings Volume 10876 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

We studied the necessary time for cell necrosis on myocardial cells against a photodynamic reaction using talaporfin sodium with various drug contact time in vitro. We have developed a new methodology of cardiac catheter ablation for tachyarrhythmia using a photodynamic reaction. To realize an immediate electrical conduction block within a few minutes, we intended to occur the photodynamic reaction outside the cells by using a short drug contact time. In a clinical situation, the drug distribution around the cells changes continuously after the drug administration. To study the change of electrical conduction block performance with these drug distribution change, we measured the necessary time for myocardial cell necrosis with various drug contact time. The concentration of intracellular Ca²⁺ ion was measured by Fluo-4 AM fluorescence dye during the photodynamic reaction with excitation light of 663 nm in wavelength under a confocal microscope. The time for myocardial cell necrosis was evaluated using a decrease of Ca²⁺ ion after the cell membrane rupture. The drug contact time for myocardial cells was varied 1-60 min. We obtained that the necessary time for myocardial cell decreased until around 10 min of drug contact time, and it increased after around 20 min of drug contact time. We suggest that the drug contact time within 10-20 min would the most effective timing to minimize the necessary time for myocardial cell necrosis using photodynamic reaction since the oxidative stress during uptake process would be high.

Over the last ten years, the need for organ donors for transplants became critical due to the increased incidence of organ failure [1]. Today, the development of tissue engineering (TE) appears as the best opportunity to overcome this shortage. TE is an interdisciplinary emerging field that aims to restore and maintain human tissue functions by applying engineering and live science principles [2]. However, one of its greatest challenges is the vascularization of tissue for the transport of oxygen and nutrients to prevent cell death. Here an innovative method is proposed to answer vascularization issues and the difficulty to create blood microcapillaries constructs, with a special interest to renal microcapillaries, which allow blood filtration. A cell-bilayer covering a tubular collagen I matrix with a diameter of about 150μm was developed and treated by ultra-short pulse (USP) laser processing in order to selectively remove the collagen core to create a capillary. The precise laser treatment allows indeed for the creation of voids in the fibre-shaped construct which results in the final formation of the capillary. Firstly, experiments were carried on a 2D model of gelatine hydrogel. The hydrogel-laser interaction was parametrically investigated in order to define a window of laser process parameters allowing the creation of voids within the hydrogel. The best window of laser process parameters was then applied to the 3D cell bilayer microfibres. Confocal microscopy examination demonstrated the presence of a lumen through the collagen I matrix without extended damage to surrounding cells. Live/Dead assays were also carried to assess cell viability.

We investigate the effect of nanosecond pulsed electric fields on optical breakdown thresholds for picosecond optical pulses (6ps pulse duration at wavelength of 1064nm) in aqueous solutions. Optical breakdown thresholds are determined both with and without the application of the nanosecond pulsed electric fields in water, phosphate-buffered saline solution, and a typical buffer solution used for live cell imaging. As optical breakdown at this time-scale mostly on a combination of multi-photon ionization as well as free electrons in the focal volume to achieve an avalanche or cascade ionization phenomena, we hypothesize that the application of a strong external electric field will have a significant impact on the breakdown probability at the lower-end of pulse energy levels, effectively modifying the pulse-energy breakdown threshold of the materials. Single optical pulses were triggered at specific times with respect to the leadingedge of the applied electric field to determine if there were any time-dependent effects on breakdown thresholds. Results of the experiments are examined and the physical phenomena as well as the concept “electrostatic purification” are discussed.

Identifying diseases and evaluating tissue function and viability can be performed by subjective or objective methods. However, subjective techniques may be inaccurate and non-optical objective techniques may be relatively expensive and time-consuming. Then, these techniques may not be suitable for clinical applications that require immediate assessment and intervention. Fluorescence spectroscopy (FS) is one of the optical techniques with great potential for medical diagnostics and surgical guidance. This potential is associated to the possibility of label-free techniques biochemical sensitivity without contrast agents. For clinical applications, fluorescence can be used to assess biomolecular content of respiratory metabolism involving NAD(P)H and FAD. In addition, changes in collagen, elastin, porphyrin, pyridoxine, and tryptophan content can potentially be detected. One way to collect epifluorescence signals from superficial tissue layers is using ultraviolet (UV) excitation. In this study, we used UV excitation FS to investigate the effect of temperature variation (from 0 to 25 degrees Celsius) on tissue autofluorescence. The measurements reproducibility was assessed by variations of the spectral shape accounted by the calculation of the Pearson correlation coefficient for each pair of measurements. Overall, fluorescence measurements were more reproducible at 25°C compared to 0°C. Liver showed lowest fluorescence variability (most homogeneous organ) regarding results from both 300 nm and 340 nm excitations. We report temperature and wavelength-dependent spectral changes due to the tissue thawing by calculating the difference between normalized UVEFS measurements at 0°C and 25°C. Observed differences may be attributed to blood absorption and NADH fluorescence emission. Our results can be used to increase the database of tissue fluorescence spectra using UV excitation for future reference to choose targeted wavelengths in fluorescence instrumentation. Furthermore, our study illustrates expected fluorescence variations during the assessment of organs viability for transplantation, especially due to cold preservation.

The mechanical properties of cells are important parameters in medicine and natural science. In this work we present a new setup that is capable of stretching adherent cells with light. For the first time, the mechanical properties of adherent cells can be determine with an active method without influencing the results by interaction of a probe or having to alter the biochemistry of the cells (e.g. by applying trypsin to detach them from the substrate). Additionally, a method to detect the resulting deformation has been developed as well as the necessary data analysis algorithms. To quantify and compare the deformation, data are fitted to viscoelastic models consisting of differently connected networks of springs and dashpods. The Akaike information criterion is used to select the best models. With the determined parameters, the mechanical properties can be assessed and 3T3 fibroblasts measured as cultured are compared to latrunculin treated ones. Regarding all parameters, the new technique delivers results in the expected range with respect to the overall mechanical properties of the cells. Furthermore, by investigating the behavior of individual parameters, also conclusions about different timescales can be drawn and the interplay of parts of the cytoskeleton during mechanical deformation is resolved. In addition, the new, active stretching technique proved to be more accurate and sensitive than the well established technique of passive microrheology.

The pathlength distribution of reflected photons can be determined from reflectance as a function of absorption coefficient through inverse Laplace transform (LT). We numerically evaluate a method based on Stehfest’s algorithm for inverse LT using exact solutions from diffusion theory. Inverse LT of the steady state reflectance equation matches the time(~pathlength) resolved reflectance equation for the same geometry, provided that the absorption coefficient is excluded from the definition of the diffusion coefficient. Different boundary conditions applied to the same measurement geometry lead to different solutions for steady state reflectance, and we investigate the effect of these changes on the extracted path length distribution. We proceed to validate the method using Monte Carlo simulations in which the reflectance as well as the pathlength of all simulated photons is stored. We confirm that the Stehfest algorithm is susceptible to noise, and introduce modifications to mitigate these effects. Knowledge of the path length distribution may be helpful in drafting models for sub-diffuse reflectance measurements such as Single Fiber Reflectance Spectroscopy, whereas moments of the pathlength distribution (mean and variance) may provide diagnostic information by themselves.

A new method for non-invasive determination of optical parameters (absorption and reduced scattering coefficients) of biological tissues from diffuse reflectance measurements with a multifiber probe is presented. For extraction of the parameters, we use a priori estimations not only of signals of detectors, but also their parameter derivatives that increases accuracy of the extraction. This estimation is performed using a new non-analog Monte Carlo algorithm which allows us to calculate the signals and derivatives simultaneously for a given large set of optical parameters. Experimental testing has shown the measured coefficients provide a good prediction of both light reflection and penetration. Application of the method to some biological tissues are presented.

Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. A label-free multimodal system combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) via a single fiber-optic interface was used for evaluation of biochemical and structural properties of tissue-engineered articular cartilage in a murine model of cartilage maturation. Nude mice (n=5) received 2 dorsal subcutaneous tissue-engineered cartilage implants each consisting of: 1) latent transforming growth factor-beta1 (LAP) treated; and 2) untreated control (CTL) constructs. At 6 weeks post-implantation, mice were sacrificed and multimodal imaging was performed in situ. FLIm showed clear delineation of the implant in all spectral bands (SB). Quantification of the cartilage construct fluorescence lifetime (LT) showed a lower LT in SB-1 (375-410 nm) and higher SB-3 LT (515-565 nm) as compared to the surrounding muscle tissue. Comparison between treatment groups showed a significant increase in FLIm SB-3 LT in LAP-treated constructs over CTL (p < 0.01). Quantification of OCT images allowed implant morphology and 3D volume comparisons between treatment groups. These results suggest that FLIm-OCT based tools are a potential non-destructive method for quantitatively monitoring the growth and quality of tissue engineered articular cartilage. The use of optical techniques to monitor maturation could represent a significant element in reducing costs in research, meeting the FDA regulatory requirements for manufacturing, and providing novel diagnostic tools in the clinic.

Optical tags have been proposed in the past for optical communication between far objects. The two-way optical link is established when the laser beam from the source reaches the optical tag, gets modulated by it and then reflected back to the source. Each optical tag should ideally have a phase conjugator for reversing the direction of the optical rays along the same path. This property dramatically enhances the coupling and the signal to noise ratio of the system in situations where the source and the optical tag are not on the line of sight of each other. In a turbid medium, the effect of scattering and phase change requires more in-depth studies. Here, we investigate this topic both experimentally and theoretically. As for the latter, we use transmission matrix approach (TMA) for full-wave solution of electromagnetic wave propagation and retroreflection in the turbid medium. In particular, we consider homodyne detection systems which rely on interferometric effects to eliminate the background stray rays and boost the signal to noise ratio. In this talk, we present our results to demonstrate that unlike flat reflectors, the retroreflectors eliminate the angular sensitivity up to 80 degrees of rotation. Whenever possible, we refer the one by one relation between the numerical simulations, theoretical analysis, and the experiments to pinpoint the origin of this enhancement. We also discuss the effect of the retroreflector size on the observed enhancement. Our results reveal the importance of retroreflectors for unprecedented signal enhancement for the emerging biomedical and atmospheric applications.

Diffuse optical imaging (DOI) with near-infrared light can reconstruct the spatial distribution of changes in absorption associated with local functional activation of the brain. Most existing studies utilize optical parameters obtained from ex-vivo measurements for the reconstruction. In consideration of the discrepancy in optical properties between individuals and especially in patients with abnormal conditions in the brain, experimental procedures were designed to quantify the optical parameters of each layer in a four-layered human head model: the scalp, skull, cerebrospinal fluid, and cerebral cortex. A near-infrared spectroscopy system combined four probes that covered 12 different source-to-detector separations (SDS) in the range of 0.215-32.4 mm for multiple depth resolved measurements. Shallower layers were measured with smaller SDS and extracted optical properties were used as initial estimates for later quantification of all four layers using larger SDS. Calibration was performed by measuring spectra of phantoms of known optical parameters and comparing to corresponding simulated spectra using the Monte Carlo method. Feasibility was validated on a three-layered solid phantom with errors in extracted optical coefficients below 15%. Finally, the prefrontal area of the human head was measured and scattering and absorption coefficients of each layer were extracted by iterative spectral fitting to Monte Carlo simulation results. Optical properties extracted from in-vivo measurements fell within reasonable ranges of reported values. In contrast to ex-vivo measurements, the presented procedure enables the reconstruction of distributions of absorption changes in the cortex using subject’s own optical properties, widening the application of functional imaging for example to post-stroke patients.

Building long lived test phantoms that simulate the scattering characteristics of biological tissue is needed in for testing optical methods targeted at imaging through tissue. Test phantoms are needed as real tissue samples optical characteristics (scattering coefficients µs and anisotropy factor g) varies greatly between samples and change rapidly with time. Our ongoing work has created long term stable phantoms with lifetimes of more than 6 years and maintaining consistent optical characteristics which mimic skin characteristics. These stable test phantom are created by an intralipid-infused agar layers 1 to 6 mm thickness Varying the intralipid concentration allows control of the scattering parameters with typical values of µs = 20/cm, g = 0.95. By encapsulating the intralipid-infused agar within a clear polymer it stabilizes for long lifetimes and allows creation a varying thicknesses, scattering characteristics and shapes. To characterize these test phantom we developed an enhanced technique where the scattered light from a laser beam passing through the test phantom is captured using a 36x24mm digital camera sensor to capture. This gives over 6 million measurements over a +/- 12 degree range, with typically 20,000 measurements at 2300 angular bins of 0.005 deg. For analysis these measurements of scattering values at a wide range of angles used a Matlab program to identify the scattering center and the angular positions. Fitted scattering models extracted the µs and g parameters for each test phantom Consistent results were obtained using a Henyey-Greenstein two-term model, probably because the Agar and intralipid impacted the scattering separately.

Computational models are useful tools for simulating the thermal response of biological tissue to laser exposure. These models typically include a solution to a bio-heat equation and require a set of physical properties for each distinct tissue as inputs. In applications such as cauterization and surgery, the irradiated tissues may undergo severe heating, resulting in substantial denaturation and a probable change in one or more of their associated physical properties. While the wellestablished temperature-dependent behavior of water can approximate the changes in tissue thermal properties during heating, there is minimal available research on the dynamics of optical properties of tissue. This study characterized optical absorption and scattering of porcine skin tissues that had undergone temperature increases up to 90°C. We excised thin samples of porcine dermis and subcutaneous fat, placed them in a custom sealed tissue mount with a built-in temperature sensor, and raised them to various temperature intervals through submergence in a controlled hot water bath. Following heating, a series of goniometric spectrophotometry measurements of transmittance and reflectance, focusing on the near-infrared band, allowed for calculation of optical absorption and scattering coefficients as a function of tissue temperature and rate process model damage. The results allow for more accurate representation of tissue characteristics in computational models.

Optical scattering properties are important diagnostic indicators with sensitivity to sub-resolution tissue structure as well as being necessary parameters for modeling light transport. Improved understanding of scattering in tissue is essential for optimizing and interpreting image contrast, modeling optogenetics, and developing next-generation optical imaging approaches. Despite the importance of optical scattering properties, most measurement methods rely on approximations or assumptions about the shape of the angular distribution of scattered light (phase function) or lack the spatial resolution to characterize heterogeneous tissue. There is a need for a spatially resolved method to quantify the optical scattering properties including the shape of the phase function. This work presents a lens-based spectral goniometry system and spatially resolved measurement of 4pi optical scattering phase functions. Angle-space measurement of scattering is performed by imaging the Fourier plane of a high-NA microscope objective. By combining forward and backward images and varying the illumination beam angle, the entire 4pi phase function can be acquired. This method enables several capabilities: a) Spatially resolved measurement of properties combined with stage scanning provides mapping of layered or heterogeneous tissues with <100 micron sampling. b) By inverting the angular scattering measurements, this approach allows characterization of refractive index autocorrelation. c) As a camera and lens based measurement technique that collects large solid-angles of scattering in a single image, the non-axially symmetric scattering signature of fibrous or oriented tissue can be characterized. These applications as well as instrument design and analysis methodology will be presented.

Low frequency Raman spectroscopy is a highly sensitive and non-destructive technique used to investigate the vibrational and rotational modes of biological and non-biological materials. The Raman spectra measured provide information about the chemical structure and nature of these materials. In this study, we present the design and construction of a low frequency Raman spectroscopy system that is able to measure signals <10 cm-1 to <400 cm-1. The system consisted of a 514.5nm monochromatic laser directed through a polarizing beam cube and half waveplate to adjust the intensity of the beam. The beam was expanded and reflected off a 514.5 nm high pass filter before passing through a 50x Mitutoyo objective, which focuses it onto the sample. The back scattered light was recollimated through the objective. The high pass filter and three 514.5 nm Bragg filters were used to reduce the Rayleigh signal. The remaining Raman signal was focused into a Shamrock 303i spectrometer with a cooled ANDOR CCD camera. Using high dynamic range data acquisition with background subtraction, this system allowed low frequency Raman spectroscopy of reduced cytochrome C, bovine serum albumin, microtubules and collagen in solution. The system has the advantage of enabling the measurement of the low frequency Raman signal without sacrificing the ability to perform traditional Raman spectroscopy.

Understanding the optical properties of water is critical to both laser-tissue interactions as well as setting ocular laser safety standards. The nonlinear properties of water are responsible for supercontinuum generation; however, these effects are poorly understood for wavelengths longer than 1064 nm. A previous study suggested that the supercontinuum generation may convert retinal-safe femtosecond near-infrared pulses with wavelengths longer than 1064 nm into visible wavelength pulses that are above the maximum permissible exposure limit as defined by ANSI Z136.1-2014. To address this knowledge gap, we extend the Z-scan technique in distilled water to wavelengths between 1150 nm to 1400 nm, where linear absorption is strong. Utilizing wavelength tunable, nominally 100 fs laser pulses, we observe wavelength dependence of the nonlinear optical properties of water. The nonlinear refractive index at 1150 nm was consistent with measurements taken at 532 nm in previous studies, and was observed to increase at longer wavelengths. The nonlinear absorption was positive for wavelengths between 1150 nm and 1350 nm and reversed to saturable absorption at 1400 nm. Saturable absorption poses a previously unanticipated eye safety risk as current ocular laser safety standards assume strong absorption at 1400 nm. These results expand our current understanding of the nonlinear optical properties of water to wavelengths in the 1150 nm to 1400 nm region, and inform efforts to revise national and international exposure limits to account for retinal hazards due to nonlinear effects.

Recent developments in high-energy regenerative amplifiers and broadly tunable optical parametric amplifiers (OPA) opened new spectral windows to study the impact of ultrashort laser pulses on biological tissues. These sources can generate extraordinarily high peak power capable of causing laser-induced breakdown. However, current laser safety standards (ANSI Z136.1-2014) do not provide guidance on maximum permissible exposure (MPE) values for the skin with pulse durations less than one nanosecond. This study measured damage thresholds in excised porcine skin in the mid-infrared (MIR) region of the electromagnetic spectrum. The laser system, comprised of a high-energy regenerative amplifier and OPA, was tuned to wavelengths between 4000-6000 nm to coincide with heightened absorption for both water and collagen. The laser operated at a fundamental repetition rate of 1 kHz and a nominal pulse width of 150 fs. The beam was focused at the sample surface with a 36X aluminum reflective objective and scanned over a 4 mm2 area for each exposure condition. Spectral domain optical coherence tomography (SD-OCT) imaging of the tissue provided a volumetric assessment of tissue morphology and identified changes in the backscattering profile within the laser-exposed regions. The determination of laser damage thresholds in the MIR for ultrafast lasers will guide safety standards and establish the appropriate MPE levels for exposure to sensitive biological tissue. These data will help guide the safe use of ultrafast MIR lasers in emerging applications across a multitude of industries and operational environments.

The use of light to inactivate microbes as an alternative method to the traditional methods of controlling microorganisms continues to draw the attention of researchers. Traditional methods of sterilization and/or pasteurization using chemicals or thermal treatments have certain limitations such as the creation of resistant bacterial strains. The application of pulsed laser irradiation compromises the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. This study aimed at using a range of power densities to investigate inactivation of Escherichia coli and Salmonella enteritidis. A Titanium sapphire pulsed laser at 800 nm wavelength, repetition rate of 76 MHz, pulse duration of 120 fs, output power of 560 mW was used in this study. A fluence range was applied on bacterial cultures in a 16 mm diameter petri with a beam spot area of 2.5 cm² (after expansion). The laser killing effectiveness was evaluated by comparing colony forming units (CFUs) with and without irradiation on 10^-7 dilutions of bacterial cultures. Cytotoxicity was analysed using the lactose dehydrogenase (LDH) assay. The laser killing rate varied with bacteria species or strains and the level of fluence.

Light interaction with live cells can lead to important effects. Near infrared (NIR) lasers are used in therapies to diminish inflammation and pain. In a microscopic scale, NIR laser light has been used to stimulate and guide the growth of cells like neurons and fibroblasts. However, it is still unclear what NIR laser radiation provokes to the cells at the molecular and biophysical levels. In this contribution we report the effects of a continuous wave 810-nm laser on the plasma membrane of in vivo 3T3 fibroblast cells. Membranes were labelled the lipophilic styryl dye FM 4-64 and imaged by confocal microscopy. We found that the NIR laser produces an increase of the fluorescence intensity at the location of laser spot. This intensity boost vanishes when the laser is turned off. The time taken by the increase and decrease is of the order of 10 seconds. The mean fluorescence increase, calculated over 75 independent measurements, equals 19%. The experiments reveal that the fluorescence rise is a growing function of the laser power. This dependence is well fitted with a square root function. The NIR laser provokes a rise in the number of molecular associations dye-lipid. The results reported here may be a consequence of a combination of induced increments in membrane fluidity and exocytosis. To the best of our knowledge, this is the first demonstration of the influence of focused NIR laser on the lipid dynamics of a live cell plasma membrane.

Identification and analysis of laser-induced lesions on the retina can be challenging in both the research and clinical settings depending on the age of a lesion and the imaging modality used for detection. Previous research exploring retinal damage thresholds utilized the consensus of an expert panel to confirm energies required for minimal visible lesions, a method that includes some subjectivity. Because of this, there is a desire to develop an image processing architecture to accurately locate retinal laser lesions in images generated from clinically relevant modalities. Issues such as imaging aberrations inducing circular artifacts, perceived stretch in lesions, and differences in the appearance of lesions across the dataset preclude use of traditional image processing tools. A database containing images of laser lesions has been developed in order to provide a reference for researchers and clinicians. In this work, we explored using various Convolutional Neural Network (CNN) architectures and preprocessing techniques to more objectively identify and analyze retinal laser lesions. Specifically, we developed frequency domain filtering techniques in order to emphasize lesion qualities. We consider this task to be one of image segmentation to make the networks somewhat size invariant. Since the lesions account for a small amount of the image pixels, we implemented an intersection-based loss function. We evaluated the performance of our trained networks against more complicated architecture variants. Additionally, we trained a network to segment and classify lesions as the result of photochemical, photomechanical or photothermal damage.

Exposures to non-ionizing electromagnetic (EM) waves in the radiofrequency (RF) range have been shown to influence gene expression in various cell and tissue types. However, the specific mechanism(s) by which exposure to these waves alter gene expression is not completely clear. Recent studies have suggested changes in epigenetics as a plausible mechanism for the gene expression alterations observed in response to exposures to RF waves. In this study, we investigated if exposures to RF fields can influence epigenetics. Specifically, we examined modifications in DNA methylation patterns in response to exposures to 900 MHz RF fields in primary human keratinocytes. We assembled a custom system to allow the stable exposure of cell cultures to 900 MHz RF fields at a range of applied powers and resultant E fields. We used methylation sensitive restriction enzyme digestion and Global DNA Methylation ELISA assay to quantify the status of global DNA methylation in cells exposed to 900 MHz RF fields for different time durations and power densities. Results show significant changes in global DNA methylation in the RF exposed cells compared to the sham (unexposed) counterparts. Importantly, these changes occur in the absence of cell death and without a concomitant increase in temperature during exposures, suggesting that alterations in DNA methylation are not associated with toxic or thermal effects of the RF fields. This suggests that RF exposure changes DNA methylation patterns and can potentially alter gene expression.

Polarized light microscopy and polarimetry has been used to assess changes in cervical structure by targeting its collagen. 75% of the human cervix is in fact composed of highly arranged collagen which is birefringent. Recently we have used Mueller Matrix polarimetry to image the human cervix in-vivo to determine loss of collagen arrangement associated with later stages of pregnancy. In an effort to improve our system capability and better determine the provenance of the polarized signal we have developed a Polarized Light Monte Carlo model capable of characterizing polarized light interaction with a birefringent, scattering, and absorbing medium such as the cervix. We have utilized this model to investigate the effect of cervical collagen arrangement typical of late stage in pregnancy on polarized light. In this talk we will illustrate the model framework, its validations, and provide several test cases in model cervices.

Biomedical optical techniques of treatment, characterization and surgery are strongly dependent on light propagation in tissues. Information that goes beyond pure intensity, such as polarization or other coherence parameters, can provide increased contrast. This contrast is critical in clinical applications, as malignant tissue has to be distinguished from healthy one, or a particular component or structure has to be highlighted and detected. The appropriate consideration of these further light-tissue interaction properties requires taking into account phase and coherence. The complexity of the problem increases as biological tissues present usually high scattering. This fact greatly influences optical propagation, and is usually a fundamental limitation in optical diagnostic techniques. Light propagation in static scattering media can be analyzed by Green’s functions. Electromagnetic propagation could be then considered, including coherence phenomena. However, analytical solutions are complex and require usually numerical methods to obtain a result. Monte Carlo approaches are particularly well-suited in biological tissues. In this work light propagation in highly scattering biological tissues is analyzed first by Green’s functions. The limited geometry of this analytical approach serves as a first approach for more complex structures. More realistic biological tissue models are proposed and solved via a threedimensional time-resolved Monte Carlo approach. The model is applied to dermatological tumoral tissues. The results of scattering by Green’s functions and the Monte Carlo approach are compared, and the potential contrast of coherence parameters is analyzed in diagnostic applications.

To calculate the light propagation through heterogeneous bio-tissues, we propose a convolutional deep network with specified convolutional kernels and calculation rules. The form of convolution kernels is chosen to be capable to imitate the absorption and scattering event by convolution operation. In the meanwhile, the multi kernel and masking mechanism we set provide the capability to calculate propagation in voxelized heterogeneous bio-tissue structures with pre-set tissue types and specific optical properties. The two-dimensional convolution operations are carried out multiple times until all the photons leaves the structure or absorbed by the tissues. Application of our network with kernels in the form of semi-infinite homogeneous radiative transfer equation (RTE) solution, and semi-infinite homogeneous diffusion equation (DE) solution are implemented to three artificial manipulated structures, including homogeneous phantom, two-layer structure and two-layer structure with a third tissue type inside layer two. The result comparing to Monte-Carlo simulation reveals the potential to form a new forward calculation model for diffuse optical tomography.

Two families of new non-analog algorithms of the Monte Carlo method are suggested for calculation of linear characteristics of optical radiation field in turbid heterogeneous media, biological tissues, first of all. One of the families is specifically aimed at calculating the readings of small-aperture radiation detectors. The algorithms allow us with high differential accuracy simultaneously for a large set of the media with different optical parameters (including phase function ones) to calculate the readings of detectors and their derivatives with respect to the parameters. This makes the algorithms high perspective for solving inverse problems of biomedical optics, in particular, for determination of optical parameters of biological tissues from measurements of radiation characteristics. The result is obtained using a rigorous approach based on the theory of Monte Carlo method for linear integral equations. For this purpose, we wrote (in the framework of the kinetic model) the adjoint integral representations for linear radiation characteristics in a heterogeneous turbid medium, correctly considering the reflection and refraction of the light on the surfaces of refractive index discontinuity.

While there exist many Monte Carlo (MC) programs for solving the radiative transfer equation (RTE) in biological tissues, we have identified a need for an open-source MC program that is sufficiently user-friendly for use in an education environment, in which detailed knowledge of compiling or UNIX command-line cannot be assumed. Therefore, we introduce MCmatlab, an open-source codebase thus far consisting of (a) a fast 3D Monte Carlo RTE solver and (b) a finite-element heat diffusion and Arrhenius-based thermal tissue damage simulator, both run in MATLAB. The kernel for both of these solvers are written in parallelized C and implemented as MATLAB MEX functions, combining the speed of C with the familiarity and versatility of MATLAB. We present example results generated by the RTE solver and the thermal model. MCmatlab is easy to install and use and can be used by students and experienced researchers alike for simulating tissue light propagation and, optionally, thermal damage.

To detect small-scale changes in tissue, small sampling volumes and, therefore, short source–detector separations are required. In this case, reflectance measurements are not adequately described by the diffusion approximation. It has been shown that such subdiffusive measurements are sensitive to the phase function of tissue (the probability distribution of scattering angles). Three parameters related to the tissue phase function have been proposed to describe subdiffusive reflectance: gamma, sigma, and RpNA. For an overlapping source-detector geometry (e.g. Single Fiber Reflectance spectroscopy, or SFR), it has been shown that RpNA outperforms gamma and sigma. RpNA was derived using the assumptions that detected photons undergo only a single backscatter event in combination with an arbitrary number of forward scattering events, and that all scattering angles occur within the acceptance angle of the detector, theta_NA, where theta_NA = arcsin(NA/nsample). We further investigated the phase function influence, by determining the distribution of scattering angles for detected photons – which we term the ‘effective phase function’. We will show that the assumption that all scattering angles occur within the acceptance angle of the detector is incorrect. Based on our results for the effective phase function, we derived a new parameter, Rpeff. We performed Monte Carlo simulations for overlapping source/detector geometries for a range of phase functions, reduced scattering coefficients, NAs, and source/detector diameters, which showed that Rpeff improves the prediction of the measured reflectance compared to gamma, sigma, and RpNA. We developed a model for an overlapping source-detector geometry incorporating Rpeff, to derive scattering and absorption properties of tissue.

Identification and analysis of laser-induced lesions on the retina can be challenging in both the research and clinical settings depending on the age of a lesion and the imaging modality used for detection. Previous research exploring retinal damage thresholds utilized the consensus of an expert panel to confirm energies required for minimal visible lesions, a method that includes some subjectivity. Because of this, there is a desire to develop an image processing architecture to accurately locate retinal laser lesions in images generated from clinically relevant modalities. Issues such as imaging aberrations inducing circular artifacts, perceived stretch in lesions, and differences in the appearance of lesions across the dataset preclude use of traditional image processing tools. A database containing images of laser lesions has been developed in order to provide a reference for researchers and clinicians. In this work, we explored using various Convolutional Neural Network (CNN) architectures and preprocessing techniques to more objectively identify and analyze retinal laser lesions. Specifically, we developed frequency domain filtering techniques in order to emphasize lesion qualities. We consider this task to be one of image segmentation to make the networks somewhat size invariant. Since the lesions account for a small amount of the image pixels, we implemented an intersection-based loss function. We evaluated the performance of our trained networks against more complicated architecture variants. Additionally, we trained a network to segment and classify lesions as the result of photochemical, photomechanical or photothermal damage.

Single-mode thulium fiber laser (TFL) at 1.94 μm with optimal energy and pulse settings has potential benefits for lithotripsy over the presently used Ho:YAG laser. A fiber Bragg grating-based, all-fiber, continuous-wave and modulated TFL at 1.94 μm is configured to deliver up to 30 W of laser power with efficiency of 50%. The TFL operating in the range of repetition rate 10 Hz-1 kHz and corresponding pulse energy 2 J-1.05 mJ is irradiated on urinary stone for in-vitro evaluation of fragmented particle size and retropulsion. TFL irradiation at higher repetition rate fragments the stones into smaller particle size (average size of few hundreds microns) resulting reduced retropulsion.

Flashlamp pumped Erbium lasers are successfully used clinical for soft and hard tissue ablation. Especially for soft tissue ablation the limited repetition rate is a disadvantage (bleeding; perforation instead of cutting). Now diode pumped solid state (DPSS) Er:YAG laser systems (Pantec Engineering AG) are available, with mean laser power up to 50 W and pulse repetition rate up to 2 kHz. The aim of this study is to investigate the potential of this laser system for increased and defined soft tissue coagulation/manipulation at various irradiation parameters, in particular at repetition rates exceeding 100 Hz. Firstly, an appropriate experimental set-up was realized with laser system, focusing unit, computer-controlled linear stage with sample holder and shutter unit to move the sample (fresh chicken breast) with a defined velocity while irradiation by various laser parameters. While irradiation the tissue effects were recorded by a video camera, adapted on a surgical microscope. After irradiation, the samples were analyzed by light microscopy. In addition, histological sections were prepared and microscopically analyzed. Mainly depending on the fluence, the thermal effect can be limited to coagulation without carbonization. In addition, tissue melting can be observed. The coagulation depth increases with increasing pulse repetition rate and decreasing movement velocity from about 30 µm to above 1 mm. In conclusion, the results of the in vitro studies show that the diode pumped, pulsed Er:YAG laser has the potential to provide one system both, for high efficient hard and soft tissue ablation as well as for soft tissue coagulation and fusion.

Conference Presentation for "30 years of Laser-Tissue Interaction and beyond"

Understanding free-electron mediated effects of tightly focused femtosecond pulse series is essential for minimizing photodamage in nonlinear microscopy and opens new avenues for nanosurgery and intentional modifications of biomolecules. We tracked different stages of the photomodification kinetics (hyperfluorescence, plasma luminescence, bubble formation) by time-lapse 2-photon microscopy, fluorescence lifetime measurements, and bubble interferometry with nanometer resolution. Monitoring of bubble growth during pulse series enabled us to quantify chemical reaction rates leading to gas formation via molecular disintegration. Novel ways of data evaluation were used to create a comprehensive picture of the photomodification kinetics in the (irradiance/irradiation dose) parameter space.

Conference Presentation for "Blood, sweat, and light (no tears!): from treatment of vascular disorders to cancer detection"

Curcumin is a natural and biocompatible compound that has been used for a variety of medical applications. These applications include treatment of several tumor cells, skin diseases, wound healing, and inflammation. Moreover, curcumin has potential to be used for theranostic of neurodegenerative diseases involving formation of Aβ plaques, as it can stain amyloid-β (Aβ) plaques and slightly improve the cognitive function in elderly. However, the diagnosis contrast and the treatment efficiency curcumin can provide are dependent on its molecular microenvironment, as it can change curcumin physical and chemical properties. In this paper, we characterize these properties for two types of curcumin formulations and suggest a quantum yield approach to enhance the detection of Aβ plaques with one of these formulations. The first formulation is synthetic curcumin (100% curcumin) and the second is Sigma Aldrich curcumin has 94% of curcuminoid content and 80% curcumin. Our measurements show that solutions containing only curcumin provided highest fluorescence signal with relatively lower optical densities, i.e., an increase of 73.2% (375 nm excitation) and 55% (445 nm excitation) in the quantum yield for the concentration of 20 μg/ml (54.30 μM). This suggests the synthetic curcumin formulation may be more efficient when used as a biomarker for diagnostics purposes or monitoring the efficiency of curcumin treatments using fluorescence spectroscopy.

In the experiment, the features of laser interstitial thermotherapy (LITT) with wavelengths of 0.92; 0.97; 1.06; 1.56 and 1.9 μm and the possibility of estimating photothermal interactions by means of ultrasoundwere studied. Ultrasound clearly determined the tissue coagulation zone without differentiating it from carbonation. The phenomenon of "breakdown" of tissues during LITT was revealed. Most often at 1.9 Μm-LITT there was a rapid transfer of heat by gas bubbles through the vessels. Intima of the vessels was fired. Ultrasound control of LITT in 145 of 781 children with vascular tumors increased its effectiveness, reducing the number of repeated sessions by 3.2 times.

Salmonella enterica is a gram-negative pathogen with great relevance in the food industry, related to food gastroenteritis. Staphylococcus aureus is a gram-positive bacterium that causes intestinal infection, vomiting, and diarrhea when its toxins are ingested. One way to combat these pathogens is photodynamic inactivation (PDI), which consists of the interaction of three elements: photosensitizer, light and molecular oxygen. This interaction promotes the formation of reactive oxygen species, which leads to cell death through necrosis and apoptosis. The present study was divided into four groups: control, light, photosensitizer, and PDI, in order to evaluate the effect of the individually applied and combined elements. In addition, a comparison between the responses of different types of bacteria was performed. The photosensitizer used was protoporphyrin IX, produced through its precursor aminolevulinic acid (ALA). The ALA was applied at concentrations of 0.4 and 0.7 μM, and incubation time from one to four hours. Irradiation was performed at 630 nm and a fluence of 12, 36 and 72 J/cm² . The results indicated that the photosensitizer concentrations were inefficient to cause total cell death, however, gram-positive bacteria were more susceptible to the technique. Photodynamic antimicrobial therapy is an alternative to existing techniques, presenting a great cost-benefit and having the advantage of being ecologically correct since it only involves non-toxic elements to cause the total destruction of the bacteria.

Current methods of teaching tissue optics in biophotonics and biomedical optics courses include creating computer-based learning environments. However, this method assumes students are going to learn or have prior experience on computer programming, which may generate time-consuming activities of several days. Then, the same activities and material of the schools and courses cannot be used in workshops or outreach activities of several hours for generating students interest in biomedical optics. This is partially compensated by websites such as omlc.org, which provide online material about biophotonics fundamentals and time flexibility to access tissue optics tools. On the other hand, students are still required to spend more time to understand tissue optics concepts and those in their first contact with biophotonics may require additional user-friendly tools. With this in mind, user-friendly tools for quick comprehension of tissue optics concepts have potential to accommodate students with diverse backgrounds and improve biomedical optics education. In this study, we designed a tissue-optics computer app that requires no previous programming experience. The app was designed to cover tissue optics topics in short length activities and generate students interest in the biomedical optics field. This computer app was tested in a 1.5 hour session within a 7-hour biophotonics workshop. By the end of the workshop, we collected students feedback about the quality of subject matter and teaching in the computer lab. Our results suggested that our app is user-friendly and is suitable for short activities. We provide a link to access the current version of our app. In the future, the app can be used in outreach activities and workshops for improvement of teaching and learning in tissue optics.

Tooth whitening treatment is a very common dentistry application for appearance enhancement. The importance of teeth appearance and color has attracted industrial interest in developing many products for consumers and professional users. However, standardizing a protocol for application of these products is difficult due to a high biological variability of the teeth color and structure. This creates a need for an objective method for monitoring of teeth shades. Current objective evaluation comprises colorimetric, spectroscopic, and imaging analysis, mostly based on reflectance and fluorescence techniques. Previous studies monitor total fluorescence intensity for this purpose, since it can take into account the effect of fluorescent agents in dental tissues. On the other hand, few spectroscopic studies investigate the processes that generate the variation in the fluorescence intensity due to staining or teeth whitening. These processes can be understood by using fluorescence spectroscopy, which has potential to be used with fluorescence imaging techniques to improve the assessment of teeth shade. This study aims to use fluorescence spectroscopy as a tool to assess tooth whitening, especially using a recently developed light-induced method using violet LED illumination. Our results show a decrease of 37.4% in the total fluorescence intensity between 450 nm and 750 nm after coffee staining. After the whitening treatment, this intensity increased 11% compared to the emission before staining. Observed spectral changes suggest the changes due to the whitening method used in this study. Also, this illustrates the potential of light-induced whitening for minimally or non-invasive whitening.