Study of the efficacy of 5 ALA-mediated photodynamic therapy on human larynx squamous cell carcinoma (Hep2c) cell line

5-aminolevulanic acid (ALA), a precursor of Protoporphyrin IX, was evaluated as an inducer of photodamage on Hep2c, human larynx squamous cell carcinoma, cell line. Porphyrins are used as active cytotoxic antitumor agents in photodynamic therapy (PDT). The present study evaluates the effects of photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) using human larynx cells as experimental model. Hep2c cell line was irradiated with red light (a diode laser, λ = 635 nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of Hep2c cells were studied. The optimal uptake of photosensitizer ALA in Hep-2c cells was investigated by means of spectrometric measurement. Cells viability was determined by means of neutral red assay (NR). It was observed that sensitizer or light doses have no significant effect on cells viability when studied independently. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 7 h in vitro incubation. The photocytotoxic assay showed that light dose of 85 J/cm² gives effective PDT outcome for Hep2c cell line incubated with 55 μg/ml of 5-ALA with a conclusion that Hep2c cell line is sensitive to ALA-mediated PDT.     

The role of sensitivity of ALA (PpIX)-based PDT on Human embryonic kidney cell line (HEK293T)

Present study evaluates the effects of photodynamic therapy (PDT) with aminolevulinic acid (5-ALA) as photo sensitizer using Human embryonic kidney (HEK293T) cell line as an experimental model. Porphyrins derivatives are used as active cytotoxic antitumor agents in PDT. Above mentioned cell line were irradiated with red light (a diode laser, λ = 635 nm) at different doses (0–160 J/cm²) of light. The influence/effectiveness of incubation time, various concentrations of aminolevulinic acid (5-ALA) and light doses on the cellular viability was studied. HEK293T cells were deliberated by exposing the ALA-PpIX (0–1000 μg/ml) of concentrations. The optimal uptakes of photosensitizer (PS) in cell lines were investigated by means of spectro photo metric measurements. Cells viability was determined by means of neutral red assay (NRA). It was observed that alone, neither photosensitizer nor light dose have significant effect on cells viability, but optimal concentration of PS along with suitable dose of light exhibit effective impact on the viability of cell. Our results showed that light doses of 40 J/cm² demonstrates effective PDT outcome for HEK293T cell line when incubated with 400 μg/ml, with wrapping up view that HEK293T cell line is very sensitive to ALA-mediated PDT as compared to cell line published in our data. At the end results has been verified by using reactive oxygen species (ROS) measure test.     

Cytotoxic and photocytotoxic effect of Photofrin® on human laryngeal carcinoma (Hep2c) cell line

Significant apoptotic effect in Hep2c cell line has been investigated, when diode laser (λ = 635 nm, red) are used as a source of illumination and initiation of photodynamic action. The optimal uptake time of Photofrin® for Hep2c cell line was investigated by means of spectrophotometric measurement. Quantification of the live cell population was determined by means of neutral red assay (NRA). The spectrometry measurements showed that after 46 h incubation, the maximal cellular uptake of Photofrin® was achieved and photocytotoxic assay showed that light dose of 120 J/cm² give effective PDT outcome for Hep2c cell line incubated with 85 μg/ml of Photofrin®. No significant phototoxic and cytotoxic effects on Hep2c cells were observed due to light doses or photosensitize, when studied independently of each other and Photofrin® showed good anti tumor effects.     

Photodynamic damage (PDD) study using stimulated raman scattering

In the current study, photodynamic damage in different cell cultures was examined using a pulsed laser light. Two different experiments were performed to analyse the photodynamic damage. For the first one, a stimulated Raman scattering laser has been obtained by exciting DMSO liquid with Nd-YAG laser, second harmonic generation, 532 nm. The resulted SRS wavelength is pulsed 630 nm. 1 ml ALA (200 μg/ml) was added to cell suspension and keep it for incubated for 4 h then irradiate the suspension with SRS wavelength 630 nm at different light dose 8, 12, 17, 25, 30, 40, 60, 100, 150, 200 μJ for 10 pluses and obtain the cell degradation. We repeat the step above but for 30 pluses. Finally for the second experiment, 1 ml ALA (200 μg/ml) was added to cell suspension and was incubated for 4 h and then irradiated with Nd-YAG Laser at wavelength 532 nm. Different doses range between 8 up to 200 μJ for 10 pluses only and the cell degradation rate was measured.     

Photodynamic damage study of HeLa cell line using ALA

The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425–525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.     

Photodynamic therapy of non-melanoma skin cancers

In this prospective study duly approved from Institutional Ethics Review Committee for research in medicine, PAEC General Hospital Islamabad, Pakistan, we investigate the efficacy, safety and tolerability along with cosmetic outcome of topical 5-aminolaevulinic acid photodynamic therapy for superficial nonmelanoma skin cancers (NMSCs) and their precursors. Patients with Histological diagnosis of NMSCs and their precursors were assessed for PDT, after photographic documentation of the lesions and written consent, underwent two (2) sessions of PDT in one month (4 weeks) according to standard protocol. A freshly prepared 20% 5-ALA in Unguentum base was applied under occlusive dressing for 4–6 h as Drug Light Interval (DLI) and irradiated with light of 630 nm wavelength from a diode laser at standard dose of 90 J/cm². Approximately 11% patients reported pain during treatment which was managed in different simple ways. In our study we regularly followed up the patients for gross as well as histopathological response and recurrence free periods during median follow-up of 24 months. Regarding Basal cell carcinomas complete response was observed in 86.2% (25/29), partial response in 10.3% (3/29) and recurrence during first year in 3.5% (1/29) lesions. All the lesions which showed partial response or recurrence were nBCCs. Regarding Actinic Keratosis complete response was observed in 95.3% (20/21), partial response in 4.7% (1/21) while Bowen’s disease showed 100% (2/2) results. 81.8% (9/11) Squamous Cell Carcinomas showed complete, 9% (1/11) partial response and 9% (1/11) presented with recurrence after 3 months. We observed excellent and good cosmetic results along with tumor clearance in our study. Treatment sessions were well tolerated with high level of patient’s satisfaction and only minor side effects of pain during treatment sessions and inflammatory changes post photodynamic therapy were observed. We concluded that 5-ALA PDT is an effective and safe emerging treatment modality for management of superficial non-melanoma skin cancers and their precursors with better cosmetic outcome and minor side effects.     

Increased viability of odontoblast-like cells subjected to low-level laser irradiation

Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm² were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO₂ at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm² + 5% FBS; G2: 1.5 J/cm² + 10% FBS; G3: 5 J/cm² + 5% FBS; G4: 5 J/cm² + 10% FBS; G5: 19 J/cm² + 5% FBS; G6: 19 J/cm² + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm². These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.     

Efficient J=0-1 soft-X-ray lasing in neon-like ions at pump powers below 250 GW

2 . By using a 0.7% prepulse that precedes the main pulse by 5 ns and applying a total pump energy of 100 J or less, the J=0-1 lasing is at least one order of magnitude higher than the non-lasing background. For the 32.6-nm line of Ti, the 25.5-nm line of Fe, and the 23.1-nm line of Ni, gain coefficients of (±) 4.20.4cm^-1, (±) 3.90.3cm^-1, and (±) 3.60.6cm^-1, respectively, were measured for 2.4-cm-long curved targets, resulting in gain–length products of ∼10. Angle-resolved spectra indicate a beam divergence of 3 mrad (FWHM), typically. The space-resolved spectra show that the J=0-1 lasing lines are emitted from an approximately 60-μm-wide (FWHM) plasma region, whereas the nearby continuum emission is produced in a considerably broader plasma region of ∼250 μm. Lasing at 25.5 nm in neon-like iron was observed at a pump power as low as 180 GW (∼9 TW/cm²), with, however, considerable shot-to-shot scatter in the absolute laser output. Received: 5 September 1997/Revised version: 10 November 1997     

Ytterbium doped all-fiber-path all-normal dispersion mode-locked laser based on semiconductor saturable mirror

In this paper, we describe a compact all-fiber-path picosecond pulse based on Ytterbium doped fiber oscillator. A home-made novel SESAM mounted on fiber is reported, by which stable mode locking is obtained. The SESAM possesses the low saturation flux 20 μJ/cm² (versus prior low saturation flux 32 μJ/cm²), which effectively reduces the pump power threshold of mode locking. The fiber laser generates 15 ps pulse trains without a dispersive delay line or anomalous dispersion in the cavity. Mode locking pulse with 30 MHz basic repetition-rate was produced, with 10–30 mW scale average output power at 1064 nm. Through 60 h of uninterrupted laser operating, mode locking is steady as ever.     

Effectiveness of Photogem® activated by LED on the decontamination of artificial carious bovine dentin

The aim of this study was the evaluation of the effectiveness of photodynamic therapy on the decontamination of artificially induced carious bovine dentin, using Photogem® as the photosensitizer agent and an LED device as a light source. Dentin samples obtained from bovine incisors were immersed in sterile broth supplemented by Lactobacillus acidophillus 10⁸ colony formation units (CFU) and Streptococcus mutans 10⁸ CFU. Different concentrations of photosensitizer, PA = 1 mg/ml, PB = 2 mg/ml, and PC = 3 mg/ml, and two fluences, D = 24 J/cm² and D = 48 J/cm², were investigated. After CFU counting per milligram of carious dentin and statistical analysis, we observed that the photodynamic therapy (PDT) parameters used were effective for bacterial reduction in the in vitro model under study. The best result was achieved with the application of Photogem® at 2 mg/ml and photoactivated under 24 J/cm² showing a survival factor of 0.14. At higher photosensitizer concentrations, a higher dark toxicity was observed. We propose a simple mathematical expression for the determination of PDT parameters of photosensitizer concentration and light fluence for different survival factor values. Since LED devices are simpler and cheaper compared to laser systems, it would be interesting to verify their efficacy as a light source in photodynamic therapy for the decontamination of carious dentin.     

Near IR laser light visualizators using nonlinear GaSe and other layered crystallites

Two methods of preparation of the devices for visualization of pulsed and continuous near-IR (near infrared) are described and the results of conversion of pulsed and continuous IR (800–1360 nm) laser radiation into the visible range of spectra (400–680 nm) by using a transparent substrate covered with the particles (including nanoparticles) of effective nonlinear materials of GaSe _x S_{1 − x} (0.2 ≤ x ≤ 0.8) are presented. Converted light can be detected in transmission or reflection geometry as a visible spot corresponding to the real size of the incident laser beam. Developed device structures can be used for checking if the laser is working or not, for optical adjustment, for visualization of distribution of laser radiation over the cross of the beam and for investigation of the content of the laser radiation. Low energy (power density) limit for visualization of the IR laser pulses with 2–3 ps duration for these device structures are: between 4.6–2.1 μJ (3 × 10⁻⁴−1 × 10⁻⁴ W/cm²) at 1200 nm; between 8.4–2.6 μJ (4.7 × 10⁻⁴−1.5 × 10⁻⁴ W/cm²) at 1300 nm; between 14.4–8.1 μJ (8.2 × 10⁻⁴–4.6 × 10⁻⁴ W/cm²) at 1360 nm. Threshold damage density is more than 10 MW/cm² at λ = 1060 nm, pulse duration τ = 35 ps. The results are compared with commercially existing laser light visualizators.     

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1×10⁵ to 1×10¹⁰ W/m²). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×10² W/m² being sufficient, provided that a total fluence of ∼30 J/cm² is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm² resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.     

Pulsed laser ablation and deposition of silicon

A KrF laser was used to ablate a polycrystalline Si target for deposition of Si on MgO and GaAs substrates at room temperature. The deposition was performed in 10⁻⁸ mbar, with two types of laser beams: a homogeneous beam being imaged onto the target (2.9 J/cm²), and a non-homogeneous which is nearly focused (2 J/cm², 6.5 J/cm²). In both cases, the beam was scanned over an area of 1 cm². For the homogenous beam, we observed only a limited number of droplets (<0.1 μm). A high number of micron-sized (<5 μm) droplets were observed on the film by the higher fluence nonhomogeneous laser beam. Raman spectroscopy showed that the micron-sized droplets are crystalline while the film is amorphous. The generation of the large droplets is most likely related to the cone structures formed on the ablated target. We also compared cone formation on a polycrystalline Si target and a single crystalline Si wafer, using multiple laser pulses onto a single spot.     

Patterning of PLZT and PSZT thin films by excimer laser

The patterning of lanthanum-doped lead zirconate titanate (PLZT) and strontium-doped lead zirconate titanate (PSZT) thin films has been examined using a 5-ns pulsed excimer laser. Both types of film were deposited by rf magnetron sputtering with in situ heating and a controlled cooling rate in order to obtain the perovskite-structured films. The depth of laser ablation in both PSZT and PLZT films showed a logarithmic dependence on fluence. The ablation rate of PLZT films was slightly higher than that of PSZT films over the range of fluence (10–150 J/cm²) and increased linearly with number of pulses. The threshold fluence required to initiate ablation was ∼ 1.25 J/cm² for PLZT and ∼ 1.87 J/cm² for PSZT films. Individual squares were patterned with areas ranging from 10×10 μm² up to 30×30 μm² using single and multiple pulses. The morphology of the etched surfaces comprised globules which had diameters of 200–250 nm in PLZT and 1400 nm in PSZT films. The diameter of the globules has been shown to increase with fluence until reaching an approximately constant size at ≤ 20 J/cm² in both types of film. The composition of the films following ablation has been compared using X-ray photoelectron spectroscopy and energy-dispersive X-ray spectroscopy. PACS 79.20.Ds; 82.80.Pv; 82.80.Ej     

Tritium recovery from co-deposited layers using 193-nm laser

Recovery of tritium from co-deposited layers formed in deuterium–tritium plasma operations of the TFTR (Tokamak Fusion Test Reactor) was investigated by the use of an ArF excimer laser operating at the wavelength of 193 nm. At the laser energy density of 0.1 J/cm², a transient spike of the tritium-release rate was observed at initial irradiation. Hydrogen isotopes were released in the form of hydrogen-isotope molecules during the laser irradiation in vacuum, suggesting that tritium can be recovered readily from the released gases. In a second experiment, hydrogen (tritium) recovery from the co-deposited layers on JT-60 tiles that had experienced hydrogen-plasma operations was investigated by laser ablation with a focused beam of the excimer laser. The removal rate of the co-deposited layers was quite low when the laser energy density was smaller than the ablation threshold (1.0 J/cm²), but reached 1.1 μm/pulse at the laser energy density of 7.6 J/cm². The effective absorption coefficient in the co-deposited layers at the laser wavelength was determined to be 1.9 μm^-1. The temperature of the surface during the irradiation at the laser energy density of 0.5 J/cm² was measured on the basis of Planck’s law of radiation, and the maximum temperature during the irradiation decreased from 3570 K at the initial irradiation to 2550 K at the 1000th pulse of the irradiation. Received: 5 August 2002 / Accepted: 7 August 2002 / Published online: 28 October 2002 RID="*" ID="*"Corresponding author. Fax: +81-29/2825917, E-mail: shu@tpl.tokai.jaeri.go.jp     

3.5-μm high-resolution gas sensing employing a LiNbO3 QPM-DFG waveguide module

Diode laser technology coupled with a wavelength-conversion unit to produce mid-infrared narrow bandwidth laser light applicable to trace-gas detection and with the potential for high-resolution spectroscopy is described. Quasi-phase-matched difference-frequency generation (QPM-DFG) in a compact and fibre-coupled periodically poled lithium niobate (PPLN) waveguide module mixing 1063 and 1525-nm radiations has been adopted for generating 34 μW of 3.5-μm wavelength laser light. Optical detection methods, including sensitive wavelength modulation spectroscopy and a rapid wavelength chirp technique, have been employed with a single-pass cell to investigate methane and formaldehyde absorption profiles around 2855 cm⁻¹, as proof of principle experiments for high sensitivity and resolution spectroscopy on atmospherically important molecules.     

Micro-lens arrays generated by UV laser irradiation of doped PMMA

Micro-lenses with well-defined optical parameters are generated on polymethylmethacrylate (PMMA) substrates doped with diphenyltriazene (DPT) by controlled use of a swelling effect generated under conditions of subablative excimer laser illumination. The surface profiles depend on the laser spot size and energy density. A sensitively balanced combination of matrix softening, substrate volume expansion due to photochemical nitrogen release, and surface tension is responsible for the final shape of the lenses. Complete arrays of identical lenses with 15 μm diameters and a focal length of 30 μm are produced by irradiation of (0.25 wt. %) DPT-PMMA with a single laser pulse at a wavelength of 308 nm and a fluence of 3 J/cm². It is shown experimentally and theoretically that appropriate volume expansion is possible without introducing internal light scattering due to the formation of small bubbles. Received: 7 April 1999 / Accepted: 8 April 1999 / Published online: 5 May 1999     

Sensitizer localization and immune response in photodynamic therapy of B16 cells

This paper proposes to extend the exploration of mouse melanoma B16 cells death by photodynamic therapy (PDT), under irradiation with different light sources and in the presence of 5,10,15,20-tetrap-sulphonato-phenyl-porphyrin (TSPP). The viability studies showed that B16 mouse melanoma is sensitive to photodynamic damage induced by TSPP 1 mM for either one, two, three or four hours. The control had TSPP added immediately prior to timelapse imaging (no incubation). They were then irradiated with red light He-Ne laser (λ = 632.8 nm, energy 180 J/cm² for 20 min). Also, it has been used a laser diode GaInAs 25 mW/cm², λ = 650 nm. The cells demonstrated clear morphological changes associated with apoptosis by mitochondrial pathway. There were changes in texture, as expected. Changes appeared to occur more quickly at lamp irradiation than at HeNe and GaInAs diode laser. Addition of TSPP just prior to exposure and observation, with no incubation, did not result in changes in cell morphology or cell death. Also, the proteins changes have been observed, because those with high molecular weights have been scissored under irradiation and this could be reason of the proteins concentrating in the area of low molecular weights, and the dissapearing of the proteic band of 75 kDa in the electrophoregramm. The immunized animals with B16-TSPP had the highest survival rate (40 days) by comparison with the control (death at 20 days) or with immunized animals with supernatants B16 (death at 25 days).     

Waveguides and quasi-waveguides based on pyrromethene 597-doped poly(methyl methacrylate)

The characteristics of amplified spontaneous emission (ASE) from asymmetric planar waveguides and quasi-waveguides consisting of thin films of poly(methyl methacrylate) incorporating lasing dye pyrromethene 597 deposited onto quartz and glass substrates, respectively, are investigated. The variable stripe length and moving constant stripe methods, together with appropriate theoretical expressions which take into account gain saturation and a simple model based on a four-level laser, allow for obtaining the net gain coefficients as a function of pump intensity, losses, pump thresholds for the onset of ASE, effective stimulated emission cross sections, pump saturation intensities, and saturation lengths. Net gain coefficients of up to 84±3 cm⁻¹ at a pump intensity of 404 kW/cm² (28 μJ/pulse) for quasi-waveguides and up to 59±6 cm⁻¹ at a pump intensity of 360 kW/cm² (25 μJ/pulse) for waveguides were obtained, with pump thresholds of 15.7 kW/cm² (1.1 μJ/pulse) and 6.3 kW/cm² (0.43 μJ/pulse), respectively. When waveguides 8 μm thick were irradiated with pulses of 200 kW/cm² at 10 Hz repetition rate, the ASE remained at 79% of its initial value after 1000 pump pulses in the same position of the sample. In quasi-waveguides 10 μm thick, the emission remained at 82% of the initial value under the same conditions.     

Resonant infrared pulsed laser deposition of thin biodegradable polymer films

Thin films of the biodegradable polymer poly(DL-lactide-co-glycolide) (PLGA) were deposited using resonant infrared pulsed laser deposition (RIR-PLD). The output of a free-electron laser was focused onto a solid target of the polymer, and the films were deposited using 2.90 (resonant with O-H stretch) and 3.40 (C-H) μm light at macropulse fluences of 7.8 and 6.7 J/cm², respectively. Under these conditions, a 0.5-μm thick film can be grown in less than 5 min. Film structure was determined from infrared absorbance measurements and gel permeation chromatography (GPC). While the infrared absorbance spectrum of the films is nearly identical with that of the native polymer, the average molecular weight of the films is a little less than half that of the starting material. Potential strategies for defeating this mass change are discussed. Received: 22 August 2001 / Accepted: 23 August 2001 / Published online: 17 October 2001