Wavelength- and irradiance-dependent changes in intracellular nitric oxide level.

SIGNIFICANCE: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O2 instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production. AIM: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells. APPROACH: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light. RESULTS: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate. CONCLUSIONS: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

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.

Microfluidic approach for direct and uniform laser irradiation to study biochemical state changes on Jurkat-T cells.

We investigated the potential for using polydimethylsiloxane microfluidic devices in a biological assay to explore the cellular stress response (CSR) associated with hyperthermia induced by exposure to laser radiation. In vitro studies of laser-tissue interaction traditionally involved exposing a monolayer of cells. Given the heating-cooling dynamics of the cells and nutrient medium, this technique produces a characteristic "bulls-eye" temperature history that plagues downstream molecular analyses due to the nonuniform thermal experience of exposed cells. To circumvent this issue, we devised an approach to deliver single cells to the laser beam using a microfluidic channel, allowing homogeneous irradiation and collection of sufficient like-treated cells to measure changes in CSR after laser heating. To test this approach, we irradiated Jurkat-T cells with a 2-µm-wavelength laser in one branch of a 100-µm-wide bifurcated channel while unexposed control cells were simultaneously passing through the other, identical channel. Cell viability was measured using vital dyes, and expression of HSPA1A was measured using reverse transcription polymerase chain reaction. The laser damage threshold was 25 ± 2 J/cm2, and we found a twofold increase in expression at that exposure. This approach may be employed to examine transcriptome-wide/proteome changes and further comparative work across stressors and cell types.

Ocular laser exposure incident reporting.

BACKGROUND: Advances in laser technology are providing opportunities for new laser applications. Once the nearly exclusive province of scientists and the military, lasers are finding increasingly widespread applications in modern life. Along with this increase in applications, it is reasonable to expect a concomitant increase in the number of accidents and occasions of purposeful misuse, resulting in excessive ocular exposures to laser radiation. METHODS: A survey was conducted of databases currently collecting laser exposure and injury information. Those found were then evaluated for content, organization, and ease of use. RESULTS: A number of laser injury databases, privately and publicly funded, were found Overall, information on laser-associated injuries was quite fragmented, showing significant variability in format and content. CONCLUSIONS: Public health policy development and clinical management of eye injuries would benefit from a single, comprehensive, and easily accessible database/information tool such as the U.S. Army's Laser Accident and Incident Registry.