Damage integral and other predictive formulas for nonisothermal heating during laser exposure.

SIGNIFICANCE: Physics-based models supply simulated temperature rises to photothermal damage rate models and provide comprehensive risk assessments for laser-induced damage. As the physics-based models continue to be refined, the damage rate models have not advanced. This peculiar lack of improvement is counterintuitive considering the damage integral (Ω), originally derived for isothermal heating events, and fails to accurately represent the nonisothermal heating from short laser exposures. AIM: Derive a nonisothermal form of the damage integral and predict more accurately the damage induced by short laser exposures, as well as identify the role of heating rate in laser damage. APPROACH: From first principles, we derived a version of the damage integral specific to the shape of thermal profiles rather than the square function provided by Arrhenius plots. We used previously published threshold thermal profiles, where all nonisothermal frequency factors (Anon) solved all Ωnon values to unity. Nonisothermal correction factors correct isothermal Aiso values. RESULTS: The Ea values were identical for both the isothermal and nonisothermal conventions. Correction factor values for Ωiso ranged from 0.0 (20-s exposures at thermal steady state) to -0.93 (0.05-s exposures). Based on empirical results, we have derived a two-dimensional empirical formula that predicts the heating rate as a function of exposure duration and ambient temperature. Threshold peak temperatures (Tpthr) and threshold critical temperatures are mathematically determined without thermal profiles when appropriate Ea and Anon values are established. CONCLUSIONS: We have identified a modified damage integral that does not rely on the Arrhenius plot and provides a value for the frequency factor (A) that accounts for the nonisothermal nature of short laser exposures. The method, validated in our in vitro retinal model, requires thermal profiles recorded under threshold conditions, such as at minimum visible lesions or the boundary of cell death. The method is a new option for laser damage modelers.

Mammalian complex III heme dynamics studied with pump-probe spectroscopy and red light illuminations.

The electronic or molecular mechanisms that initiate photobiomodulation (PBM) in cells are not yet fully understood. The porcine complex III (C-III) of the electron transport chain was characterized with transient absorption spectroscopy (TAS). We then applied our recently developed continuous wave laser coupled TAS procedure (CW-TAS) to investigate the effect of red light irradiances on the heme dynamics of C-III in its c1 reduced state. The time constants were found to be 3.3 ± 0.3 ps for vibrational cooling of the oxidized state and 4.9 ± 0.4 ps for rebinding of the photodissociated axial ligand of the c1 reduced state. The analysis of the CW-TAS procedure yielded no significant changes in the C-III heme dynamics. We rule out the possibility of 635 nm CW light at 4.7 mW/cm2 inducing a PBM effect on the heme dynamic of C-III, specifically with the photodissociation of its axial ligand.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Transient absorption spectroscopy to explore cellular pathways to photobiomodulation.

Photobiomodulation (PBM) describes the use of low irradiance light in the red to near-infrared wavelength range to stimulate biological effects in tissue, and many biological and spectroscopic techniques are used to study PBM. However, these techniques focus on the products or downstream effects rather than the electronic transitions that initiate the PBM processes. This study presents a novel approach to studying low irradiance light exposures on individual proteins and/or protein complexes by combining a continuous wave (CW) laser diode with femtosecond transient absorption spectroscopy (TAS), coined here as CW-TAS, and tests the system on reduced cytochrome c (Cyt c) for proof of principle. TAS was conducted using a 532-nm excitation pump beam and a 350-600 nm supercontinuum probe. CW laser diodes with wavelengths of 450 nm, 635 nm, and 808 nm were interchangeably fiber coupled into the HELIOS Fire. Samples of Cyt c were tested by TAS using a pump power of 15 µW, both with and without CW exposure. CW exposures were carried out with irradiances of 1.60 and 3.20 mW/cm2, except for 808 nm, which was only tested at 1.60 mW/cm2. Both kinetic and global analyses were performed on the TAS data and the time constants for sets with and without CW exposures were compared. The TAS data for Cyt c with the full dosage of CW exposures did not alter the TAS data distinguishably from the control data. No new electronic transient signals were observed beyond the background when testing Cyt c with the CW exposures. Kinetic analysis confirmed that existing transients did not deviate beyond uncertainty. Global time constants for Cyt c were calculated to be 0.25 ± 0.03 ps and 5.1 ± 0.3 ps for the control study, and the time constants for the CW exposed Cyt c were not significantly different. This study concludes that CW irradiation, at doses delivered, does not alter the transient absorption data of Cyt c. The CW-TAS method provides a new tool for studying PBM effects in other proteins and protein complexes that might respond to the CW wavelengths, such as Complex IV, in future studies.

Quantitative phase microscopy monitors subcellular dynamics in single cells exposed to nanosecond pulsed electric fields.

A substantial body of literature exists to study the dynamics of single cells exposed to short duration (<1 µs), high peak power (~1 MV/m) transient electric fields. Much of this research is limited to traditional fluorescence-based microscopy techniques, which introduce exogenous agents to the culture and are only sensitive to a single molecular target. Quantitative phase imaging (QPI) is a coherent imaging modality which uses optical path length as a label-free contrast mechanism, and has proven highly effective for the study of single-cell dynamics. In this work, we introduce QPI as a useful imaging tool for the study of cells undergoing cytoskeletal remodeling after nanosecond pulsed electric field (nsPEF) exposure. In particular, we use cell swelling, dry mass and disorder strength measurements derived from QPI phase images to monitor the cellular response to nsPEFs. We hope this demonstration of QPI's utility will lead to a further adoption of the technique for the study of directed energy bioeffects.

Evaluation of the potential eye hazard at visible wavelengths of the supercontinuum generated by an ultrafast NIR laser in water.

Lasers with ultrashort pulse durations have become ubiquitous in various applications, including ocular surgery. Therefore, we need to consider the role of nonlinear optical effects, such as supercontinuum generation during propagation within the ocular media, when evaluating their potential hazard. We used a NIR femtosecond laser to generate a supercontinuum within an artificial eye. We recorded the visible spectra of the supercontinuum generated and calculated the energy contained within the visible band. Our results indicate that for wavelengths between 1350 nm and 1450 nm the energy contained within the visible band of the generated white light supercontinuum may surpass current safety exposure limits, and pose a risk of injury to the retina.

Continuous assessment of metabolic activity of mitochondria using resonance Raman microspectroscopy.

Dysfunctional mitochondrial activity can lead to a variety of different diseases. As such, there exists a need to quantify changes in mitochondria function as it relates to these specific diseased states. Here, we present the use of resonance Raman (RR) spectroscopy as a tool to determine changes in isolated mitochondrial activity. RR spectroscopy, using 532 nm as the excitation source, specifically provides information on the reduction and oxidation (RedOx) state of cytochrome c, which is determined by the activity of protein complexes in the electron transport chain (ETC). In this model, injection of the substrate succinate into the mitochondrial sample is used to drive the ETC, which causes a subsequent change in cytochrome c RedOx state. This change in RedOx state is tracked by RR spectroscopy. This tool gives real-time information on the rise and fall of the amount of reduced cytochrome c within the mitochondrial sample, providing a method for rapid assessment of mitochondrial metabolism that has broad applications in both basic science and medical research.

Thermal damage thresholds for multiple-pulse porcine skin laser exposures at 1070 nm.

As solid-state laser technology continues to mature, high-energy lasers operating in the near-infrared (NIR) band have seen increased utilization in manufacturing, medical, and military applications. Formulations of maximum permissible exposure limits establish guidelines for the safe use of these systems for a given set of laser parameters, based on past experimental and analytical studies of exposure thresholds causing injury to the skin and eyes. The purpose of our study is to characterize the skin response to multiple-pulsed laser exposures at the NIR wavelength of 1070 nm, at a constant beam diameter of 1 cm, using anesthetized Yucatan mini-pig subjects. Our study explores three constant total laser-on times of 0.01, 0.1, and 10 s as single- and multiple-pulse sequences. Exposures consisting of 10, 30, and 100 pulses have identical individual pulse durations but different duty cycles in order to include variable degrees of thermal additivity. A plurality of three observers quantifies skin damage with the minimally visible lesion metric, judged at the 1- and 24-h intervals postexposure. Calculation of the median effective dose (ED50) provides injury thresholds for all exposure conditions, based on varying laser power across subjects. The results of this study will provide a quantitative basis for the incorporation of multiple-pulsed laser exposure into standards and augment data contained in the existing ED50 database.

Filamentation in Atmospheric Air with Tunable 1100-2400 nm Near-Infrared Femtosecond Laser Source.

Intense femtosecond pulse filamentation in open-air has been utilized for long distance optical communication and remote sensing, but it results in nonlinear-effect driven eye hazards which are not addressed by current eye safety standards. A systematic study of filamentation in atmospheric air was performed using a tunable 100 fs near-infrared laser (1100 nm-2400 nm). While undergoing filamentation, each source wavelength was spectrally broadened resulting in supercontinuum and third harmonic generation in the visible and near-IR spectrum. We record the spectra at the center and fringes of the supercontinuum as it is imaged onto a planar surface. In a full beam collection regime, we report the energy of the sub-1000 nm light generation for source wavelengths from 1100 nm to 1600 nm and compare the energy density to the maximum permissible exposure values under the ANSI Z136.1 laser safety standard.

Effect of ambient temperature and intracellular pigmentation on photothermal damage rate kinetics.

Computational models predicting cell damage responses to transient temperature rises generated by exposure to lasers have implemented the damage integral (Ω), which time integrates the chemical reaction rate constant described by Arrhenius. However, few published reports of empirical temperature histories (thermal profiles) correlated with damage outcomes at the cellular level are available to validate the breadth of applicability of the damage integral. In our study, an analysis of photothermal damage rate processes in cultured retinal pigment epithelium cells indicated good agreement between temperature rise, exposure duration (τ), and threshold cellular damage. Full-frame thermograms recorded at high magnification during laser exposures were overlaid with fluorescence damage images taken 1 h postexposure. From the image overlays, pixels of the thermogram correlated with the boundary of cell death were used to extract threshold thermal profiles. Assessing photothermal responses at these boundaries standardized all data points, irrespective of laser irradiance, damage size, or optical and thermal properties of the cells. These results support the hypothesis that data from boundaries of cell death were equivalent to a minimum visible lesion, where the damage integral approached unity (Ω = 1) at the end of the exposure duration. Empirically resolved Arrhenius coefficients for use in the damage integral determined from exposures at wavelengths of 2 µm and 532 nm and durations of 0.05-20 s were consistent with literature values. Varying ambient temperature (Tamb) between 20°C and 40°C during laser exposure did not change the τ-dependent threshold peak temperature (Tp). We also show that, although threshold laser irradiance varied due to pigmentation differences, threshold temperatures were irradiance independent.

Suprathreshold laser injuries in excised porcine skin for millisecond exposures at 1070 nm.

Skin injury response to near-infrared (NIR) laser radiation between the minimum visible lesion threshold and ablation onset is not well understood. This study utilizes a 1070-nm diode-pumped Yb-fiber laser to explore the response of excised porcine skin to high-energy exposures in the suprathreshold injury region without inducing ablation. Concurrent high-speed videography is employed to determine a dichotomous response for three progressive damage categories: observable surface distortion, surface bubble formation due to contained intracutaneous water vaporization, and surface bubble rupture during exposure. Median effective dose (ED50) values are calculated in these categories for 3- and 100-ms pulses with beam diameters (1 / e2) of 3 mm (28, 35, and 49 J / cm2) and 7 mm (96, 141, and 212 J / cm2), respectively. Double-pulse cases are secondarily investigated. Experimental data are compared with the maximum permissible exposure limits and ablation onset simulated by a one-dimensional multiphysics model. Logistic regression analysis predicted injury events with ∼90 % of accuracy. The distinction of skin response into progressive damage categories expands the current understanding of high-energy laser safety while underlining the unique biophysical effects during induced water phase change in tissue. These results prove to be useful in the diagnosis and treatment of NIR laser injuries.

Z-scan measurements of water from 1150 to 1400 nm.

Understanding the nonlinear properties of water is essential for laser surgery applications, as well as understanding supercontinuum generation in water. Unfortunately, the nonlinear properties of water for wavelengths longer than 1064 nm are poorly understood. We extend the application of the Z-scan technique in water to determine its nonlinear refractive index (n2) and nonlinear absorption (ß) for wavelengths in the 1150-1400 nm range, where linear absorption is also significant. We observe the wavelength-dependent variation of the nonlinear properties of water around the water absorption band.

Single-shot chemical detection and identification with compressed hyperspectral Raman imaging.

Raman imaging is a powerful method to identify and detect chemicals, but the long acquisition time required for full spectroscopic Raman images limits many practical applications. Compressive sensing and compressed ultrafast photography have recently demonstrated the acquisition of multi-dimensional data sets with single-shot detection. In this Letter, we demonstrate the utilization of compressed sensing for single-shot compressed Raman imaging. In particular, we use this technique to demonstrate the identification of two similarly white substances in one image via the recovered two-dimensional array of Raman spectra. This technique can be further extended by coupling the compressed sensing apparatus with a microscope for compressed hyperspectral imaging microscopy.

Probe beam deflection optical imaging of thermal and mechanical phenomena resulting from nanosecond electric pulse (nsEP) exposure in-vitro.

Electric-field induced physical phenomena, such as thermal, mechanical and electrochemical dynamics, may be the driving mechanism behind bioeffects observed in mammalian cells during exposure to nanosecond-duration electric pulses (nsEP) in-vitro. Correlating a driving mechanism to a biological response requires the experimental measurement and quantification of all physical dynamics resulting from the nsEP stimulus. A passive and electromagnetic interference (EMI) immune sensor is required to resolve these dynamics in high strength electric fields. The probe beam deflection technique (PBDT) is a passive and EMI immune optical method for quantifying and imaging refractive index gradients in liquids and gases, both dynamic and static, with nanosecond temporal resolution. In this work, a probe beam deflection imaging system was designed to acquire 2-D time-lapse images of thermal/mechanical dynamics resulting from monopolar and bipolar nsEP stimulus.

Enabling time resolved microscopy with random Raman lasing.

Optical imaging of fast events and processes is essential for understanding dynamics of complex systems. A bright flash of illuminating light is required to acquire sufficient number of photons for superior image quality. Laser pulses can provide extreme brightness and are typically employed to achieve high temporal resolution; however, the high degree of coherence associated with the lasing process degrades the image quality with speckle formation. Random lasers are low-coherence sources of stimulated emission and do not suffer from speckle, but are rather broadband and have a relatively low output power limiting the scope of their potential applications. In this report, we demonstrate the use of random Raman lasing as a novel imaging light source with unprecedented brightness for a speckle-free and narrowband light source. We showcase the advantages of a random Raman laser to image the nanosecond scale dynamics of cavitation formation in water and quantitatively compare these images to those taken with incoherent fluorescent emission and coherent laser light as illumination source.

Trends in nanosecond melanosome microcavitation up to 1540 nm.

Thresholds for microcavitation of bovine and porcine melanosomes were previously reported, using single nanosecond (ns) laser pulses in the visible (532 nm) and the near-infrared (NIR) from 1000 to 1319 nm. Here, we report average radiant exposure thresholds for bovine melanosome microcavitation at additional NIR wavelengths up to 1540 nm, which range from ∼0.159 J∕cm2 at 800 nm to 4.5 J∕cm2 at 1540 nm. Melanosome absorption coefficients were also estimated, and decreased with increasing wavelength. These values were compared to retinal pigment epithelium coefficients, and to water absorption, over the same wavelength range. Corneal total intraocular energy retinal damage threshold values were estimated and compared to the previous (2007) and recently changed (2014) maximum permissible exposure (MPE) safe levels. Results provide additional data that support the recent changes to the MPE levels, as well as the first microcavitation data at 1540 nm, a wavelength for which melanosome microcavitation may be an ns-pulse skin damage mechanism.

Robust commercial diffuse reflector for UV-VIS-NIR applications.

We report the development and testing of a new commercially available diffuse reflecting material with reflectivities in the visible comparable to industry-leading products. This new diffuse reflector consists of solid quartz in which there is a dense distribution of tiny pockets of air. The multiple reflections by the quartz-air interfaces of these air pockets transforms a highly transmissive base material into a highly diffuse reflecting material.

Thermal evaluation of laser exposures in an in vitro retinal model by microthermal sensing.

A temperature detection system using a micropipette thermocouple sensor was developed for use within mammalian cells during laser exposure with an 8.6-µm beam at 532 nm. We have demonstrated the capability of measuring temperatures at a single-cell level in the microscale range by inserting micropipettebased thermal sensors of size ranging from 2 to 4 µm into the membrane of a live retinal pigment epithelium (RPE) cell subjected to a laser beam. We setup the treatment groups of 532-nm laser-irradiated single RPE cell and in situ temperature recordings were made over time. Thermal profiles are given for representative cells experiencing damage resulting from exposures of 0.2 to 2 s. The measured maximum temperature rise for each cell ranges from 39 to 73°C; the RPE cells showed a signature of death for all the cases reported herein. In order to check the cell viability, real-time fluorescence microscopy was used to identify the transition of pigmented RPE cells between viable and damaged states due to laser exposure.