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.

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.

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.

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.

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.

Single-shot stand-off chemical identification of powders using random Raman lasing.

The task of identifying explosives, hazardous chemicals, and biological materials from a safe distance is the subject we consider. Much of the prior work on stand-off spectroscopy using light has been devoted to generating a backward-propagating beam of light that can be used drive further spectroscopic processes. The discovery of random lasing and, more recently, random Raman lasing provide a mechanism for remotely generating copious amounts of chemically specific Raman scattered light. The bright nature of random Raman lasing renders directionality unnecessary, allowing for the detection and identification of chemicals from large distances in real time. In this article, the single-shot remote identification of chemicals at kilometer-scale distances is experimentally demonstrated using random Raman lasing.

Assessment of tissue heating under tunable near-infrared radiation.

The time-temperature effects of laser radiation exposure are investigated as a function of wavelength. Here, we report the thermal response of bulk tissue as a function of wavelength from 700 to 1064 nm. Additionally, Monte Carlo simulations were used to verify the thermal response measured and predict damage thresholds based on the response.

Bright emission from a random Raman laser.

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm(-1). Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium.

High-resolution in vivo imaging of regimes of laser damage to the primate retina.

Purpose. To investigate fundamental mechanisms of regimes of laser induced damage to the retina and the morphological changes associated with the damage response. Methods. Varying grades of photothermal, photochemical, and photomechanical retinal laser damage were produced in eyes of eight cynomolgus monkeys. An adaptive optics confocal scanning laser ophthalmoscope and spectral domain optical coherence tomographer were combined to simultaneously collect complementary in vivo images of retinal laser damage during and following exposure. Baseline color fundus photography was performed to complement high-resolution imaging. Monkeys were perfused with 10% buffered formalin and eyes were enucleated for histological analysis. Results. Laser energies for visible retinal damage in this study were consistent with previously reported damage thresholds. Lesions were identified in OCT images that were not visible in direct ophthalmoscopic examination or fundus photos. Unique diagnostic characteristics, specific to each damage regime, were identified and associated with shape and localization of lesions to specific retinal layers. Previously undocumented retinal healing response to blue continuous wave laser exposure was recorded through a novel experimental methodology. Conclusion. This study revealed increased sensitivity of lesion detection and improved specificity to the laser of origin utilizing high-resolution imaging when compared to traditional ophthalmic imaging techniques in the retina.

Trends in melanosome microcavitation thresholds for nanosecond pulse exposures in the near infrared.

Thresholds for microcavitation of bovine and porcine melanosomes were determined using nanosecond laser pulses in the near-infrared (1000 to 1319 nm) wavelength regime. Isolated melanosomes were irradiated by single pulses (10 or 50 ns) using a Q-switched Spectra Physics Nd:YAG laser coupled with an optical parametric oscillator (1000 to 1200 nm) or a continuum laser at 1319 nm. Time-resolved nanosecond strobe photography after the arrival of the irradiation beam allowed imaging of microcavitation events. Average fluence thresholds for microcavitation increased nonlinearly with increasing wavelength from ∼0.5 J/cm2 at 1000 nm to 2.6 J/cm2 at 1319 nm. Fluence thresholds were also measured for 10-ns pulses at 532 nm and found to be comparable to visible nanosecond pulse values published in previous reports. Calculated melanosome absorption coefficients decreased from 925 cm-1 at 1000 nm to 176 cm-1 at 1319 nm. This trend was found to be comparable to the decrease in retinal pigmented epithelial layer absorption coefficients reported over the same wavelength region. Estimated corneal total intraocular energy retinal damage threshold values were determined in order to compare to current and proposed maximum permissible exposure (MPE) safe levels. Results from this study support recently proposed changes to the MPE levels.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

Hyperthermia sensitizes pigmented cells to laser damage without changing threshold damage temperature.

We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65 W/cm2 at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.

Localized thermal mapping using coherent anti-Stokes Raman spectroscopy.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy is explored as a tool for obtaining micro-scale thermal measurements. A single femtosecond oscillator is used to pump a photonic crystal fiber to provide the broadband Stokes pulse. The CARS signals from the broad OH-stretching modes between 3000 and 3600 cm(-1) are shown to correlate with temperature with an accuracy of ± 1°C for water and ± 1.5°C for phosphate-buffered saline. Local variation of temperature is mapped on a microscopic level, using black-dyed microspheres as thermal sources.

Chemically Specific Imaging Through Stimulated Raman Photoexcitation and Ultrasound Detection: Minireview.

A powerful combination of chemically specific Raman excitation and deep tissue ultrasound imaging holds the promise to attain spatially resolved distribution of chemical compounds inside the scattering medium. In this report, an attempt is made to evaluate the recent achievements and possible challenges with an eye on potential clinical applications.

Stimulated Raman scattering using a single femtosecond oscillator with flexibility for imaging and spectral applications.

Stimulated Raman scattering (SRS) is a powerful tool for obtaining background-free chemical information about a material without extrinsic labeling. Background-free spectra are particularly important in the fingerprint region (~800 and 1800 cm(-1)) where peaks are narrow, closely-spaced, and may be in abundance for a particular chemical. We demonstrate a method for obtaining SRS spectra using a single femtosecond laser oscillator. A photonic crystal fiber is used to create a supercontinuum to provide a range of Stokes shifts from ~300 to 3400 cm(-1). This SRS approach provides for collection capabilities that are easily modified between obtaining broadband spectra and single-frequency images.

Monitoring stimulated Raman scattering with photoacoustic detection.

A capability of high-frequency ultrasound detection to monitor the process of energy deposition into a molecular system via Raman excitation is experimentally demonstrated. It is shown that the generated ultrasound signal is directly proportional to the optical signal generated in stimulated Raman scattering. Ultrasound detection provides a simple way to discriminate against laser-induced breakdown and allows for the quantification of the stimulated Raman scattering process where direct optical detection is not available. Additionally, it can be used for stimulated Raman imaging in deep tissue, provided that the generated photoacoustic signal is sufficiently strong.

Spatially correlated microthermography maps threshold temperature in laser-induced damage.

We measured threshold temperatures for cell death resulting from short (0.1-1.0 s) 514-nm laser exposures using an in vitro retinal model. Real-time thermal imaging at sub-cellular resolution provides temperature information that is spatially correlated with cells at the boundary of cell death, as indicate by post-exposure fluorescence images. Our measurements indicate markedly similar temperatures, not only around individual boundaries (single exposure), but among all exposures of the same duration in a laser irradiance-independent fashion. Two different methods yield similar threshold temperatures with low variance. Considering the experimental uncertainties associated with the thermal camera, an average peak temperature of 53 ± 2 °C is found for laser exposures of 0.1, 0.25, and 1.0 s. Additionally, we find a linear relationship between laser exposure duration and time-averaged integrated temperature. The mean thermal profiles for cells at the boundary of death were assessed using the Arrhenius rate law using parameter sets (frequency factor and energy of activation) found in three different articles.