Larry E. Estlack

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.

In vitro model that approximates retinal damage threshold trends

Without effective in vitro damage models, advances in our understanding of the physics and biology of laser-tissue interaction would be hampered due to cost and ethical limitations placed on the use of nonhuman primates. We extend our characterization of laser-induced cell death in an existing in vitro retinal model to include damage thresholds at 514 and 413 nm. The new data, when combined with data previously reported for 532 and 458 nm exposures, provide a sufficiently broad range of wavelengths and exposure durations (0.1 to 100 s) to make comparisons with minimum visible lesion (in vivo) data in the literature. Based on similarities between in vivo and in vitro action spectra and temporal action profiles, the cell culture model is found to respond to laser irradiation in a fundamentally similar fashion as the retina of the rhesus animal model. We further show that this response depends on the amount of intracellular melanin pigmentation.

Damage thresholds for cultured retinal pigment epithelial cells exposed to lasers at 532 nm and 458 nm

The determination of safe exposure levels for lasers has come from damage assessment experiments in live animals, which typically involve correlating visually identifiable damage with laser dosimetry. Studying basic mechanisms of laser damage in animal retinal systems often requires tissue sampling (animal sacrifice), making justification and animal availability problematic. We determined laser damage thresholds in cultured monolayers of a human retinal pigment epithelial (RPE) cell line. By varying exposure duration and laser wavelength, we identified conditions leading to damage by presumed photochemical or thermal mechanisms. A comparison with literature values for ocular damage thresholds validates the in vitro model. The in vitro system described will facilitate molecular and cellular approaches for understanding laser-tissue interaction.

Determination of threshold average temperature for cell death in an in vitro retinal model using thermography

Even though laser exposures of 1 s or less are non-isothermal events, researchers have had to rely upon the isothermal treatise of Arrhenius to describe the laser damage rate processes. To fully understand and model thermal damage from short exposure to laser irradiation we need to experimentally obtain the temperature history of exposed cells and correlate it with the cellular damage outcomes. We have recorded the thermal response of cultured retinal pigment epithelial cells in real-time with laser exposure using infrared imaging (thermography). These images were then overlaid with fluorescence images indicating cell death taken 1 hr post laser exposure. The image overlays allowed us to define the thermal history of cells at the boundary (threshold) of laser-induced death. We have found a correlation between the onset of cell death and the average temperature over the course of the laser exposure.

In-vitro Retinal Model Reveals a Sharp Transition between Laser Damage Mechanisms

We use laser damage thresholds in an in-vitro retinal model, and computational simulations to examine the laser exposure durations at which damage transitions from photothermal to photochemical at 413 nm. Our results indicate a dramatic shift in 1-h damage thresholds between exposure durations of 60 and 100 s. The trend in our in-vitro results is similar to a trend found in a recent study where retinal lesions were assessed 1-h post laser exposure in the rhesus eye Our data suggest that nonthermal mechanisms did not significantly contribute to cell death, even for exposures of 60 s. Knowledge of the transition point, and lack of concurrent thermal and nonthermal damage processes, are significant for those wishing to devise a comprehensive computational damage model.

The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

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

We were unable to reproduce published inactivation results, or show any interaction, between 90 femtosecond (fs) pulses of 850 nm or 425 nm laser radiation and buffer/water, DNA, protein, M13 bacteriophage or E. coli. Using agarose electrophoresis and polyacrylamide gel electrophoresis, we examined purified plasmid DNA (pUC19), bovine serum albumin, and DNA and coat proteins extracted from M13 following exposures to irradiances of up to 120 MW/cm2. We measured M13 viability using an assay for plaque-forming ability in soft agar after exposure to the same irradiances used for the protein and DNA experiments. Exposures of up 1 GW/cm2 at 850 nm had no effect on the viability of E. coli as measured by a colony forming assay in soft agar. Peroxynitrite, known to be toxic, to cause single strand breaks in DNA, and fragment proteins in vitro gave positive results in all assays.

An in vitro model for retinal laser damage

Ocular laser exposures resulting in damage at the retina typically involve cellular alterations in the retinal pigment epithelial (RPE) layer. To provide guidelines for eye-safe exposure to lasers, the laser safety community has relied on damage assessment in nonhuman primate studies. Simple and reliable model systems for laser bioeffects that use cultured RPE cells, rather than animals, are thus desirable. We have characterized our artificially pigmented hTERT-RPE1 model by identifying ED50 thresholds over a wide range of laser parameters and cell culture conditions. When summarized as action spectra and temporal action profiles (log threshold fluence versus log exposure duration), trends (pigment-dependent) in our cell model data are strikingly similar to the threshold trends reported for animal models (literature). In addition, the rapidity and flexibility (laser delivery) with which studies are performed in our culture model has benefited computational modeling efforts.

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

Abstract. 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.