This PDF file contains the front matter associated with SPIE Proceedings Volume 10861, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Transcranial infrared laser stimulation (TILS) refers to the use of infrared laser to photobiomodulate the human brain, which has been reported beneficial in enhancing human cognition. We previously investigated TILS-induced electrophysiological effects and observed increases of power density in alpha wave oscillation. However, clear association between the brain wave alteration and improvement of neural cognition is limited. Phase–amplitude coupling (PAC) is a recently proposed neural mechanism for coordinating information processing across brain regions. In this study, we wish to examine if TILS would create any enhanced PAC at particular frequency bands in particular brain regions. A 64-channel electroencephalography (EEG) system was employed to determine placebo-controlled, electrophysiological activities from 19 healthy human participants before, during and after TILS. After a 2-minute baseline, we applied a 1064-nm laser with a total power of 3.5 W on the right forehead of each human participant for 8 minutes, followed by a 3-minute recovery period. An EEG processing package (Brainstorm) was used to perform cross-frequency PAC analysis for each participant’s measurement, followed by group-level, paired T-tests between the placebo and TILS conditions. The statistical results showed that TILS induced significant inter-cerebral PAC among several brain oscillations, specifically (1) slow-delta (0.5-1 Hz) activity modulating both alpha band (8-11 Hz) and high gamma band (70-85 Hz) activities, and (2) alpha band (8-11 Hz) modulating high gamma band (70-85 Hz) activity. All of these results suggest that TILS is able to enhance thalamocortical, cortical-hippocampal-cortical, and hippocampal-thalamic activity, all of which lead to enhancement of human cognition.

Background and Objective: Extraoral photobiomodulation therapy (PBM Therapy) is a novel treatment for the prevention of oral mucositis, a painful side effect of myeloablative chemotherapy, and there are no standardized dosimetry protocols for this procedure. We used Monte Carlo modeling to determine optimal parameters for a safe and efficacious treatment. The objective of this work was to experimentally validate the results of Monte Carlo dose modeling of extraoral PBM Therapy by conducting a pilot validation study. Methods: Light penetration through the right cheek of four volunteers with skin types I-VI was measured. A 69-LED array with an area of 31.2 cm2 was applied to the external cheek delivering 26 mW/cm2 at 850 nm to the surface. Power density at the intraoral mucosa was recorded under a controlled pressure of 18gm/cm2. To obtain morphological information, we acquired T1 weighted MRI images of the volunteers' heads and measured the thickness of the skin, fat, and muscle layers of the cheek of each volunteer. These anatomical data together with the optical properties from the literature were used to simulate light propagation through the right cheek. Results and Conclusions: Our study revealed that experimental and simulation results were in good agreement for all 4 subjects. The difference between the mean values of the measured fluence rates was within 16% from the respective fluence rates obtained using Monte Carlo simulations. We confirmed that there was no temperature increase due to illumination. Monte Carlo modeling is a robust and reliable method for PBM Therapy light dosimetry.

Photo-neuro-modulation (PNM), or photobiomodulation therapy (PBMT) of neuronal tissue, has demonstrated stimulating axonal growth of the spinal cord after acute structural damage in rodent models. The effectiveness of PNM for rodent models of spinal cord injury has been established at terminal irradiances of approximately 3.2mW/cm2. For clinical translation however, the existing surface irradiation protocol results in significant treatment dose attenuation by the vertebral bones as the light travels to the spinal cord. We reported that with a surface CW power and irradiance as intense as 10W and 3.14W/cm2 respectively less than 100µW/cm2 irradiance reached the spinal cord level of dogs. In exploring clinical transmission of photobiomodulatory irradiance to the spinal cord level, we piloted in a cadaveric rabbit model local light delivery to the cerebral-spinal-fluid (CSF) space using a 200µmx1.75mm (diameterxlength) diffuser fiber at L6/7 via a spinal needle. The irradiances at multiple sites of up to 9 centimeters distance along the spinal canal were measured sequentially by a second diffuser fiber via the corresponding vertebrae disks. At an intra-spinal irradiation of 8mW CW 830nm light which was 1/1,250 of the 10W surface power used for the transcutaneous dosimetry in dogs, irradiances at 1.5~2cm from the local delivery site were 1~2 mW/cm2 (2 rabbits, 4 measurements). The distance-dependent irradiances along the spinal canal corresponded to an effective attenuation coefficient of 0.1021±0.0213 mm-1 (n=4). In comparison (p=0.0274), the distance-dependent irradiances along the epaxial muscle upon interstitial delivery corresponded to an effective attenuation coefficient of 0.0677±0.0105 mm-1 (n=4). “Epidural” PNM is feasible.

Photobiomodulation (PBM) is a biological outcome of exposure to low-level light in the red and near-infrared (NIR) wavelengths. Current literature has attributed beneficial effects to PBM, to include improved wound healing, enhanced mitochondrial function, functional enhancements in patients suffering from stroke, and improved cognitive function in a murine model for traumatic brain injury. Cytochrome c oxidase, also named complex IV (C-IV) in the electron transport chain (ETC), is the expected primary chromophore for the red and NIR exposures. The direct evidence that PBM is a consequence of absorption by C-IV is incomplete. Recently, our lab has found metabolic perturbations in cells and isolated mitochondria from low-level exposures to blue and green light as well. To study the immediate and early events of PBM we used a combination of fluorescence microscopy, resonance Raman spectroscopy, Fourier transformed IR (FTIR) spectroscopy, and ultrafast transient absorption spectroscopy (TAS) on cells, isolated mitochondria, and purified ETC enzymes. In this paper, we show that FTIR spectroscopy is useful in determining substrate-dependent, steady-state rates of CO₂ production by the tricarboxylic acid (TCA) cycle. The method allows for determinations of wavelength-specific changes in metabolic rate in real time with low-level light exposures. These data will help determine if any mitochondrial components have absorption spectra that correlate with the global PBM response in the literature.

Keloids scars are an abnormal overgrowth of fibrotic tissue in response to an injury. The current treatments show several limits and do not represent a definitive solution or a prevention protocol. In a preliminary study, we irradiated two samples of human keloid fibroblasts with a Blue LED light, evidencing a possible modulation of their activity in vitro. In the current study, we use primary fibroblasts cultures from eight keloid tissues (from seven selected patients undergoing aesthetic surgery). The fibroblasts were irradiated with a Blue LED light and the treatment time was varied in the range 5÷60s. After irradiation, cell metabolism and cell proliferation were studied by the use of two colorimetric tests, CCK-8 and SRB (Sigma-Aldrich, Saint Louis, Missouri, USA). The analysis was performed 24 and 48h after the treatment. We thus evidenced that the Blue LED light induces a modulation of the fibroblasts metabolism; this effect is particularly evident at 30s irradiation time. We also evaluated the impact of Blue LED light on membrane currents by performing whole-cell patch-clamp recordings. We observed a significant increase of voltage dependent outward currents activated by a depolarizing ramp-protocol upon Blue LED light irradiation (@30s exposure). This effect was maintained in K⁺ free-solutions, thus ruling out the involvement of K⁺ channels. In conclusion, we demonstrated that the Blue LED light has a photobiomodulation effect in fibroblasts from human keloids. This effect can be proposed as a possible treatment of the wound site in human patients to prevent keloid scars occurrence.

Photobiomodulation (PBM) refers to a non-destructive, non-thermal application of near infrared or infrared light (lasers or LEDs) on human tissues for medical benefits, such as for wound healing and pain reduction. The mechanism of PBM was understood as the absorption of photon energy by cytochrome-c-oxidase (CCO), the last enzyme in the mitochondrial respiratory chain that catalyzes the reduction of oxygen for energy metabolism. However, it is unclear which wavelengths would be optimal for PBM applications due to lack of systematic studies on wavelength dependence of PBM-induced effects. In this study, we wish to quantify and compare hemodynamic and metabolic benefits of PBM on human tissue with several central wavelengths. We employed 4 LEDs, whose spectral ranges were commonly used in PBM: 760 nm (FWHM=24nm), 810 nm (FWHM=30nm), 970 nm (FWHM=46nm), and 1070 nm (FWHM=55nm). The power densities of these LEDs were measured and calibrated by a spectrometer and power meter. We applied 5-minute PBM using each of these LEDs in vivo on the right forearm of 15 human participants. Corresponding placebo experiments were also carried out for rigorous comparison. Broadband near infrared spectroscopy (740-900 nm) was employed near the PBM site to quantify hemodynamic and metabolic responses during the PBM/placebo and a 3-minute recovery period. A multi-linear regression analysis based on the modified Beer-Lambert law was performed to estimate changes of oxy-hemoglobin, deoxy-hemoglobin, and cytochrome-c-oxidase concentration. Different patterns of hemodynamic and metabolic responses were observed for these 4 LED wavelengths, showing quantitative wavelength-dependent PBM effects.

More than 20% of all newborns admitted to the neonatal intensive care unit experience abnormally low platelet counts, called neonatal thrombocytopenia. Neonatal thrombocytopenia could increase a risk of hemorrhage, especially within the brain and adversely affect its development. No drugs or therapies are currently available for these sick newborns besides platelet transfusions. However, too many platelet transfusions could induce anti-platelet autoantibodies and other complications in some cases. One innovative idea for an alternative and non-invasive therapy for thrombocytopenia is low-level laser treatment (LLLT). LLLT is very safe, like blue-light-mediated treatment of newborn jaundice that has been used in clinics for decades with a long record of safety. LLLT stimulates the expression of a group of genes involved in mitochondrial generation in megakaryocytes (MKs) that are the precursor of platelets. This stimulation increases mitochondrial mass in MKs and enlarges the cells, which in turn elevates the rate of platelet production per MK. Our early investigation demonstrated that LLLT could quickly increase the number of circulating platelets and greatly reduce the risk of bleeding in thrombocytopenic adult animals. To explore a potential of this novel modality in management of neonatal thrombocytopenia, we investigated whether LLL could increase the platelet biogenesis in fetal liver and bone marrow MKs that intended to have low polyploidy compared to those MKs in adults. We found that LLL enhanced platelet production in MKs differentiated from fetal bone marrow and livers albeit at a slightly less efficiency. In addition to increased production of platelets from MKs, LLL appeared to increase the number of MKs differentiated from MK precursors in both bone marrow and liver of newborn mice in vivo as well, in contrast to little megakaryocytopoiesis induced by LLLT in adult mice. The ability of LLLT to increase not only platelet generation from MKs but also megakaryocytopoiesis in both livers and bone marrows in newborns suggests a potential for LLLT to manage neonatal thrombocytopenia and reduce the number of transfusions for thrombocytopenia-afflicted newborns.

Photodynamic therapy (PDT) is a well-established treatment modality for cancer and other malignant diseases; however, quantities such as light fluence, photosensitizer photobleaching rate, and PDT dose do not fully account for all of the dynamic interactions between the key components involved. In particular, fluence rate (Φ) effects are not accounted for, which has a large effect on the oxygen consumption rate. In this preclinical study, reacted reactive oxygen species ([ROS]_rx) was investigated as a dosimetric quantity for PDT outcome. The ability of [ROS]_rx to predict the cure index (CI) of tumor growth, CI = 1 – k/k_ctr, where k and k_ctr are the growth rate of tumor under PDT study and the control tumor without PDT, respectively, for BPD-mediated PDT was examined. Mice bearing radioactively-induced fibrosarcoma (RIF) tumors were treated with different in-air fluences (22.5, 40, 45, 50, 70 and 100 J/cm²) and in-air Φ (75 and 150 mW/cm²) with a BPD dose of 1 mg/kg and a drug-light interval of 15 mins. Treatment was delivered with a collimated laser beam of 1 cm diameter at 690 nm. Explicit dosimetry of initial tissue oxygen concentration, tissue optical properties, and BPD concentration was used to calculate [¹O₂]_rx. Φ was calculated for the treatment volume based on Monte-Carlo simulations and measured tissue optical properties. CI was used as an endpoint for four dose metrics: light fluence, photosensitizer photobleaching rate, PDT dose, and [ROS]_rx. PDT dose was defined as the product of the time-integral of photosensitizer concentration and Φ at a 3 mm tumor depth. Preliminary studies show that [ROS]_rx best correlates with CI and is an effective dosimetric quantity that can predict treatment outcome. The threshold dose for [ROS]_rx is determined to be 0.12 mM and is about 8 times smaller than the corresponding value for conventional BPD-mediated PDT using DLI of 3 hrs.

Photobiomodulation using light from red and near infrared LEDs or Lasers have been reported effective as noninvasive methods for fat loss. A total of 55 subjects were randomly divided into test groups and control groups for abdominal fat reduction clinical trial using red and near infrared LED phototherapy devices. Red and near infrared light with irradiance of 10 mW/cm² were irradiated over the abdominal area to the test group for 30 minutes at 3 times a week followed by 3 times of 30 minutes of aerobic exercise a week for 4 weeks. Control group used sham devices for 30 minutes 3 times a week and followed by 3 times of 30 minutes of aerobic exercise a week for 4 weeks. It was shown that red and near infrared LED phototherapy combined with aerobic exercise was effective and safe for abdominal fat loss without any side effects.

Photobiomodulation Therapy offers the potential to reduce post-operative and acute injury pain and inflammation and accelerate healing which has been demonstrated in the literature and through our clinical trials. One of the barriers to its widespread use has been the availability of a simple to use hands free portable light delivery system which conforms to the tissue surface and delivers appropriate levels of irradiance without excessive heat. Our team focused on the development of new roll-to-roll printed micro LED technology in combination with a cadmium-free quantum dot film to provide a combination of 450 nm blue and red 640 nm light in a flexible light bandage. The micro LEDs are printed on 125 micron film and laminated to a flexible Printed Circuit and CFQD layer. A transparent hydrogel acts as a light pipe and adhesive to hold the flexible light source to the tissue to be treated. The light patch is powered by a small Bluetooth enabled controller module making it a hands free, wire free wearable system. The patch may be placed over a transparent film dressing when used post-surgically or placed directly over intact skin over the area of soft tissue injury. Printed LED light patches in sizes ranging from 25 to 70 cm2 have been successfully developed at irradiance levels of 9 mW/cm2 in pulsed modes at 33% DF. These highly flexible light sources closely couple light to the treatment site using optical matching layers designed to optimize optical transmission to the tissue.

We performed Transient Absorption Spectroscopy (TAS) on samples of cytochrome c in its reduced and oxidized states as well as mitochondria, and we use kinetic analysis to determine the decay rates of the transients. We also tested the samples following red light exposures to determine if there were any changes in transient decay rates. Mitochondria were isolated from hTERT-RPE1 cells and were tested in a glutamate buffer solution while cytochrome c was tested in a Sodium Phosphate solution (Na₂PO₄). Femtosecond TAS was performed with a pump pulse with wavelength centered at 418 nm and a supercontinuum probe pulse with a spectrum between 440 and 760 nm. Red light illuminations were performed with a 635 nm continuous wave light source at an intensity of 1.6 mW/cm² for 0.5 hours. We find transients and kinetic decay rates for reduced and oxidized cytochrome c that are consistent with previous literature. However, we do not find any transients for mitochondria under our current TAS testing parameters, and we expect that this may be due to low Signalto- Noise ratio or choice of pump wavelength.

Photobiomodulation (PBM) describes the use of low levels of visible to near infrared light to induce positive biological effects. While there is a growing body of research demonstrating therapeutic and protective benefits of PBM, the underlying molecular mechanisms remain poorly understood. The putative chromophore for PBM effects is cytochrome c oxidase, also known as complex IV (C-IV) of the mitochondrial electron transport chain. It is believed that via action on C-IV, light absorption initiates a cascade of events involving nitric oxide (NO), reactive oxygen species (ROS), and increased mitochondrial function, leading to improved cellular robustness. However, little is known about the mechanisms by which these signaling pathways are initiated and how they establish the observed beneficial outcomes. We have conducted in vitro analyses of living human cells to probe changes in NO, ROS, and mitochondrial function, particularly focusing on the immediate and early effects of light exposure. This analysis utilizes a novel multi-channel laser assembly, integrated with a fluorescence microscope, and collimated for a sub-field-of-view beam diameter. By combining this apparatus with specific vital stains, we can visualize production of NO, ROS, and changes in mitochondrial morphology and dynamics in real-time, during and immediately following light exposure. Additionally, we are able to compare these effects to unexposed cells within the same field-of-view. Moving forward, future studies will examine additional vital stains in cells and isolated mitochondria, allowing us to distinguish direct mitochondrial effects from those requiring cellular feedback. In summary, this research provides a new window into the molecular underpinnings of the PBM response.

Human skin contains photolabile nitric oxide (NO) derivatives which decompose after UVA irradiation and release vasoactive NO. However, aside from blue light, barely nothing has been reported about the effects of red and NIR wavelengths. We decided to investigate if photobiomodulation, using visible to NIR light, would increase the release of NO in the skin. A custom-built airtight sleeve which envelopes the forearm of a subject was used to measure the NO emanating from the skin under photobiomodulation conditions and quantified by chemiluminescence detection. Distinct differences in measured NO levels were observed between the non-irradiated condition and PBM conditions.

Background and Objective: Intraoral photobiomodulation therapy (PBMT) is effective at preventing oral mucositis (OM). Extraoral PBMT is a novel approach with distinct advantages over intraoral PBMT, but no evidence-based treatment protocol has been proposed. The goal of this contribution was to develop a practical and effective treatment protocol for extraoral PBMT to prevent OM. Methods: Extraoral PBMT was modeled using Monte Carlo simulations with parameters established in intraoral PBMT: 50 mW/cm2 (1 minute treatment time), at either 660 nm, used in superficial PBMT, or 850 nm, used in deeper PBMT. The effective therapeutic dose of ~ 2.0 J/cm2 was assumed to be identical for intraoral and extraoral PBMT. The results prompted a review of the literature regarding PBMT parameters for deep tissue treatment, as well as the mechanism of PBMT. Summary: Per our Monte Carlo simulations, the treatment parameters established for intraoral PBMT are not appropriate for extraoral delivery. Visible light (660 nm) showed poor penetration, producing an insufficient dose at the oral mucosa. The infrared light (850 nm) penetrated deeper. However, at 50 mW/cm2 and 1 minute treatment time, the dose was still less than targeted 2 J/cm2. Therefore, based on the optical properties of tissues, anatomy of the target population, and established protocols for deeper PBMT, we propose a wavelength of 850 nm, at 399 mW/cm2, and exposure time of 1-11 minutes, depending on the treatment site, to achieve the therapeutic dose of ~2 J/cm2 at the depth of the oral mucosa for preventing OM using extraoral PBMT.

Photobiomodulation, also known as low level laser therapy (LLLT), is a technique that uses light in the red and near infrared (NIR) range (600-900 nm) to elicit a clinically beneficial physiological change in tissue. This physiological change is thought to begin in the mitochondria by altering the metabolic rate for the electron transport chain (ETC). Resonance Raman spectroscopy at 532 nm was used to determine the reduction/oxidation (redox) state of cytochrome c in isolated mitochondria after undergoing LLLT. Mitochondria from hTERT-RPE1 cells were isolated and placed in glutamate buffer and then exposed to violet (405 nm) or red (635 nm) light. The resonance Raman spectrum of the cytochrome c redox state before and after light illumination was measured. This gives us an insight into the types of metabolic changes that occurs within the mitochondria while being illuminated by light during photobiomodulation.

Photobiomodulation therapy (PBMT) has demonstrated efficacy in various areas of medical practice including pain management, wound healing, inflammation treatment, and treatment for neurological diseases. More recently, animal model studies have reported that near-infrared light irradiation to a spinal cord injury (SCI) site transcutaneously enhances axonal regeneration and functional recovery. Many such studies typically use a variety of irradiation parameters and surface irradiance to calculate the fluence delivered to the injury site, and many times ignoring factors such as tissue optical properties, beam divergence, beam positioning, and tissue thickness to the organ of interest. While these studies show a broad range of treatment outcomes, a comparison of treatment efficacy among these studies with respect to light fluence is many times extremely difficult. Therefore in this study, we use Monte Carlo simulation to provide an overview of the effect of the light source probe parameters and positioning on an injury site using a 3D voxelated SCI rat phantom model with regards to PBMT. In this study, an 810 nm Top-hat beam was simulated for 3 numerical apertures (NA) (0, 0.4, and 0.8), 5 beam diameters (0.04, 0.1, 0.2, 0.4, and 0.8 cm), and 14 different irradiation positions relative to the SCI injury site. Our findings are beneficial for research into understanding the effects of the probe parameters on tissues and organs, which ultimately will aid in reducing the variability in the used fluence and help optimize PBMT outcomes.

Muscle fatigue can lead to decreased performance and be a precursor to muscle injury. We determine the effects of a novel blue 450 nm and red 630 LED light patch on repeated bouts of a reliable elbow flexion fatigue protocol. Methods: We enrolled 34 strength trained individuals and determined their 1 repetition maximum (RM) for a controlled elbow flexion task using their non-dominate arm. After at least 4 days rest the participants completed a fatigue task using a weight of 50% of their 1 RM. During the fatigue task a marker was set to 90° of elbow flexion. The participant complete elbow flexion repetitions at 25 rep/min. The task was stopped when the participant was unable to move the weight back to the marker or they were in the wrong position at the metronome beat. After the fatigue task a 30-minute active red-blue or sham treatment, determined by random assignment, was applied. The active treatment had a peak irradiance of 9 mW/cm2, 33% duty cycle, and fluence of 5.4 J/cm2. After the treatment, participants repeated the fatigue protocol with the same working weight (50% of 1 RM). The number of repetitions were counted during the pre- and post-treatment fatigue tasks. Results: 29.4% of active treatment participants improved the number of fatigue repetitions while no participants in the sham treatment improved during the post-treatment fatigue task (P = 0.045). Conclusion: The use of a blue-red flexible light patch over a muscle group can reduce muscle fatigue during repetitive intense exercises.

The mechanism responsible for the oxidative stress due to photobiomodulation induced by 1265 nm laser is still unclear. Mitochondria are assumed to be the most probable acceptors of the 1265 nm laser irradiation. We study oxidative stress, mitochondrial potential, GSH, cell viability, DNA damage. We demonstrated that narrowband (highcoherent) and wideband lasers employed at the doses of 9.45 and 66.6-400 J/cm², respectively, induce a dose-dependent cell death, increase ROS level, disturb mitochondrial functioning and can damage DNA. Thus, the 1265 nm lasers can affect the HCT116 cells through mitochondrial damage. Energy density increase contributes to cell damaging without heating effects.

The clinical prospect of treating spinal cord injury by photobiomodulation therapy (PBMT) is unclear as the spinal dosimetry of transcutaneous laser application is unknown, yet essential for clinical recommendations. Light irradiances at 9 sites over an 8cm length along the thoracic to lumbar segment of the spinal canal were measured in 6 cadaver dogs using a flexible intra-spinal probe, under a surface irradiance of 3.14W/cm². The skin transmits 4-12% of 980nm PBMT light. On-contact technique is ~5 times more efficient than off-contact technique. Transcutaneous transmission of photobiomodulatory irradiance to the spinal canal will benefit from contact-probe technique and skin-clearing approaches.