Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day‐old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225 mJ/cm² induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser‐based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

     

Laser‐induced plasmon‐mediated treatment of retinoblastoma in viscous vitreous phantom

Retinoblastoma (RB) is a rare form of cancer of the retina most prevalent in young children. We successfully show that laser‐induced cell disruption, mediated by gold plasmonic nanoparticle (NP), is a potential and efficient therapy to kill the cancerous cells. The proof of concept is demonstrated in vitro on cultured Y79 RB cancer cells with a nanosecond laser at 527 nm, for both attached cells at the bottom of a Petri dish and for floating, clustered cells in a viscous vitreous phantom comprised of hyaluronan. We report a cellular death of 82% after irradiation in classic culture medium and a cellular death of 98% in vitreous phantom, for similar number of NPs in each sample. It is found that the NPs efficiently penetrate the floating Y79 clusters cells in the vitreous phantom, leading to a cellular death of over 85% even within the centre of the aggregates. The proposed treatment technique is based on a similar nanosecond laser used to eliminate floaters in the vitreous, but with much lower (100‐1000 times) fluences of 20 J cm^?2.      

Two ways to inactivate the Ki‐67 protein—Fragmentation by nanoparticles,crosslinking with fluorescent dyes

Light can manipulate molecular biological processes with high spatial and temporal precision and optical manipulation has become increasingly popular during the last years. In combination with absorbing dyes or gold nanoparticles light is a valuable tool for cell and protein inactivation with high precision. Here we show distinct differences in the underlying mechanisms whether gold nanoparticles or fluorescent dyes are used for the inactivation of the Ki‐67 protein. The proliferation‐associated protein Ki‐67 was addressed by the antibody MIB‐1. In vitro studies showed a fragmentation of the Ki‐67 protein after laser irradiation of 15 nm gold nanoparticle antibody conjugates with nanosecond pulsed laser, while continuous wave (cw) irradiation of fluorescein isothiocyanate (FITC)‐ and Alexa 488‐labeled antibodies led to specific crosslinking of Ki‐67. The irradiation energy for the gold nanoparticles was above cavitation bubble formation threshold. We observed a fragmentation of the target protein and also of the gold particles. The understanding of the underlying inactivation mechanisms is important for the application and further development of these two techniques, which can harness nanotechnology to introduce molecular selectivity to biological systems.     

Inactivation ofSaccharomyces cells by 8-methoxypsoralen plus pulsed laser irradiation in the wavelength range 308 nm-380 nm

Summary Two different strains ofSaccharomyces cerevisiae, one diploid wild type and one haploid mutant deficient in excision repair were irradiated with laser pulses in the range 308 nm to 380 nm after 8-MOP treatment. Both the shoulder (Dq) and the final slope (Do) of the inactivation curves were dependent on wavelength which showed a broad minimum around 355 nm. No differences in inactivation were recorded after pulsed irradiations between the repetition rates of 5 Hz and 35 Hz. Irradiations with pulses of the energy density from 0.1 mJ/cm² up to 26 mJ/cm² resulted in a final slope increasing with pulse energy density. This was in contrast to the effects of irradiation alone.Abbreviations 8-MOP 8-methoxypsoralen - UV ultraviolet - PUVA therapy withPsoralen plusUV-A     

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts

We investigated the biological effects of Er:YAG laser (2940‐nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm^?2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm^?2 promoted maximal cell proliferation, determined by WST‐8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post‐irradiation. Elevation of ATP level, Ki67 staining, and cyclin‐A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post‐irradiation at 6.3 J cm^?2, and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up‐regulation of 21 genes involved in heat‐related biological responses and ER‐associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real‐time PCR. Surface temperature measurement confirmed that 6.3 J cm^?2 generated heat (40.9°C post‐irradiation). Treatment with 40°C‐warmed medium increased proliferation. Laser‐induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm^?2 strongly potentiated HGF proliferation via photo‐thermal stress, suggesting potential wound‐healing benefit.      

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

Sialolithiasis: Application parameters for an optimal laser therapy

In‐vitro experimental parametric studies of laser ablation using natural sialoliths and artificial stones have been performed toward an efficient laser treatment of sialolithiasis. Surface microstructure and water adsorption become critical for coupling high power pulsed Ho:YAG laser radiation (λ = 2080 nm, τ ～250 μsec), inducing ablative interactions and stone fragmentation. Results reveal a generic trend, with single pulse laser energy density threshold for sialolith ablative erosion at ～200 J cm^?2 (corresponding to intensity ～800 kW cm^?2) and fragmentation rates reaching ～1 mm/pulse at ～2400 J cm^?2. This process shows no saturation, suggesting that very high energy density irradiation at low pulse repetition rate is an efficient approach. Such operation facilitates rapid cooling and minimal thermal loading of the oral and maxillofacial area, thus causing negligible adverse effects. The method is expected to contribute to the establishment of an easy and optimal therapeutic protocol for sialolithiasis pathology.     

The thermodynamic response of soft biological tissues to pulsed ultraviolet laser irradiation.

The physical mechanisms that enable short pulses of high-intensity ultraviolet laser radiation to remove tissue, in a process known as laser ablation, remain obscure. The thermodynamic response of biological tissue to pulsed laser irradiation was investigated by measuring and subsequently analyzing the stress transients generated by pulsed argon fluorine (ArF, lambda = 193 nm) and krypton fluorine (KrF, lambda = 248 nm) excimer laser irradiation of porcine dermis using thin-film piezoelectric transducers. For radiant exposures that do not cause material removal, the stress transients are consistent with rapid thermal expansion of the tissue. At the threshold radiant exposure for ablation, the peak stress amplitude generated by 248 nm irradiation is more than an order of magnitude larger than that produced by 193 nm irradiation. For radiant exposures where material removal is achieved, the temporal structure of the stress transient indicates that the onset of material removal occurs during irradiation. In this regime, the variation of the peak compressive stress with radiant exposure is consistent with laser-induced rapid surface vaporization. For 193 nm irradiation, ionization of the ablated material occurs at even greater radiant exposures and is accompanied by a change in the variation of peak stress with radiant exposure consistent with a plasma-mediated ablation process. These results suggest that absorption of ultraviolet laser radiation by the extracellular matrix of tissue leads to decomposition of tissue on the time scale of the laser pulse. The difference in volumetric energy density at ablation threshold between the two wavelengths indicates that the larger stresses generated by 248 nm irradiation may facilitate the onset of material removal. However, once material removal is achieved, the stress measurements demonstrate that energy not directly responsible for target decomposition contributes to increasing the specific energy of the plume (and plasma, when present), which drives the gas dynamic expansion of ablated material. This provides direct evidence that ultraviolet laser ablation of soft biological tissues is a surface-mediated process and not explosive in nature.     

Investigation of thermal distribution for pulsed laser radiation in cancer treatment with nanoparticle-mediated hyperthermia

In this paper, we have simulated the efficacy of gold/gold sulfide (GGS) nanoshells in NIR laser hyperthermia to achieve effective targeting for tumor photothermal therapy. The problem statement takes into account the heat transfer with the blood perfusion through capillaries, and pulsed laser irradiation during the hyperthermia. Although previous researchers have used short laser pulses (nanosecond and less), in order to prevent heat leakage to the neighbor tissues, we have examined the effect of millisecond pulses, as the extent of the target volume to which hyperthermia is induced is usually larger and also the lasers with this specification are more available. A tumor with surrounding tissue was simulated in COMSOL software (a finite element analysis, solver and simulation software) and also in a phantom made of agarose and intralipid. The tumor was irradiated by 10, 20 and 30 laser pulses with durations of 15, 50 and 200 ms and fluences of 20, 40 and 60 J/cm². Experimental tests performed on a phantom prove the ability of the applied numerical model to capture the temperature distribution in the target tissue. We have shown that our simulation permits prediction of treatment outcome from computation of thermal distribution within the tumor during laser hyperthermia using GGS nanoshells and millisecond pulsed laser irradiation. The advantage of this simulation is its simplicity as well as its accuracy. Although, to develop the model completely for a given organ and application, all the parameters should be estimated based on a real vasculature of the organ, physiological conditions, and expected variation in those physiological conditions for that application in the organ.     

In Vitro Laser Ablation of Natural Marine Biofilms

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 ± 27.8 μm (titanium) and 107.4 ± 28.5 μm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm²). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.     

Laser-Driven Precipitation and Modification of Silver Nanoparticles in Soda Lime Glass Matrix Monitored by On-line Extinction Measurements

In this work, we investigated the effect of nanosecond laser irradiation at 532?nm on precipitation of Ag nanoparticles (NPs) in soda lime glasses doped with silver in the Ag^?+??CNa^?+? ion-exchange process. Formation and subsequent modification of Ag NPs during laser irradiation were studied by on-line extinction measurements making use of the localized surface plasmon resonance (LSPR). These investigations were further completed using scanning and transmission electron microscopies to examine the average size and distribution of nanoparticles within the sample. It has been shown that formation of NPs, its kinetics and the particle size strongly depend on the fluence and the total number of deposited laser pulses. It has been found that Ag NPs form after some specific number of pulses and they rapidly grow in size and number until some maximal value of extinction has been reached. Further irradiation of such samples only results in destruction of precipitated NPs due to photo-breakup, laser ablation confirmed by strong plasma emission observation. Moreover, due to strong irradiation, the host matrix can also be affected by changing its refractive index which manifests as the blue shift of the LSPR.     

Laser exposure of gold nanorods can induce intracellular calcium transients

Uncoated and poly(styrene sulphonate) (PSS)‐coated gold nanorods were taken up by NG108‐15 neuronal cells. Exposure to 780 nm laser light at the plasmon resonance wavelength of the gold nanorods was found to induce intracellular Ca²⁺ transients. The higher Ca²⁺ peaks were observed at lower laser doses, with the highest levels obtained at a radiant exposure of 0.33 J/cm². In contrast, the cells without nanoparticles showed a consistently small response, independent of the laser dose. These initial results open up new opportunities for peripheral nerve regeneration treatments and for more efficient optical stimulation techniques. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin

The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm^?2 and surface energy power density 195 mW cm^?2 (1,000 Hz) and 230 mW cm^?2 (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.     

脉冲1 064 nm激光视网膜损伤动物模型的建立

目的:建立脉冲1 064 nm Nd:YAG激光致视网膜出血性损伤及非出血性损伤动物模型,为治疗药物评价提供技术基础.方法:应用自由振荡脉冲及调Q脉冲1 064 nm激光照射青紫蓝灰兔视网膜,通过在光路中加人透镜获得直径200μm眼底光斑,加入衰减片改变角膜入射激光能量.照射即刻对损伤应用检眼镜进行实时观察,并用眼底相...     

The UVB-induced synthesis of vitamin D3 and 1α,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: Effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D₃ pathway was found to be in the range of 300 ± 5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D₃ pathway. The aim of this study was to compare the vitamin D₃ and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300 ± 2.5 nm and 310 ± 2.5 nm in organotypic cultures of keratinocytes supplemented with 25 μM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm² and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300 ± 2.5 nm and 30 mJ/cm². Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.     

Nd-YAG laser irradiation damages to Verrucaria nigrescens

Epilithic and endolithic microorganisms and lichens play an important role in stone biodeterioration. The structural and physiological damage caused by nanosecond pulsed laser of 1064 nm from Nd:YAG laser to Verrucaria nigrescens lichen as well as to endolithic algae and fungi were investigated in the present study. Ultrastructural laser effects on lichen and endolithic microorganisms were study without disturbing the relationship between lichen and lithic substrate by taking lichen-containing rock fragments and processing both together. SEM-BSE, LT-SEM and FM were used to determine cell integrity and ultrastructure, which reflect microorganism viability. Photobiont vitality was determined using a PAM chlorophyll fluorescence technique. The lichen thalli were completely removed by irradiation with 5 ns pulses at a fluence of 2.0 J/cm² with no stone damage as showed by micro-Raman spectroscopy. The fungal and algal endolithic cells located below were completely destroyed or presented a high plasmolysis degree resulting from heating their microenvironment. The lichen and endolithic mycobiont near the irradiated zone were also damaged. Algal photosynthetic damage prevents fungal survival and lichen viability. This is the first report of laser removal and inactivation of lichen and lithic microorganisms, and thus provide an environmentally friendly and efficient method to control stone biodeterioration.     

Cancer cell‐specific protein delivery by optoporation with laser‐irradiated gold nanorods

The delivery of macromolecules into living cells is challenging since in most cases molecules are endocytosed and remain in the endo‐lysosomal pathway where they are degraded before reaching their target. Here, a method is presented to selectively improve cell membrane permeability by nanosecond laser irradiation of gold nanorods (GNRs) with visible or near‐infrared irradiation in order to deliver proteins across the plasma membrane, avoiding the endo lysosomal pathway. GNRs were labeled with the anti‐EGFR (epidermal growth factor receptor) antibody Erbitux to target human ovarian carcinoma cells OVCAR‐3. Irradiation with nanosecond laser pulses at wavelengths of 532 nm or 730 nm is used for transient permeabilization of the cell membranes. As a result of the irradiation, the uptake of an anti‐Ki‐67 antibody was observed in about 50 % of the cells. The results of fluorescence lifetime imaging show that the GNR detached from the membrane after irradiation.     

Emission Efficiency and Amplification Properties of the Plasma of a Pulsed Discharge in Ar at Elevated Pressures

The spontaneous emission efficiency of an ${\rm Ar}_2^*$ excimer and its amplification properties at a wavelength of 126 nm are studied using a numerical model of the weakly ionized plasma of a pulsed discharge in Ar at elevated pressures. It is shown that, under real experimental conditions, it is possible to achieve a net gain coefficient of the active medium equal to ≈0.065 cm^?1 by increasing the gas density up to 4.0 × 10²⁰ cm^?3 at an initial gas temperature of 170 K. The internal conversion efficiency of discharge energy into spontaneous emission depends weakly on the gas temperature and attains 75% for a gas density of 2.7 × 10²⁰ cm^?3, but with excitation powers much lower than for the maximum gain. The applicability of the model at low excitation powers is tested by comparison with the experimental data.