Molecule transfer into mammalian cells by single sub-nanosecond laser pulses.

A rapid, precise, and viability-retaining method for cytoplasmic molecule delivery is highly desired for cell engineering. Routine methods suffer from low throughput, lack of selectivity, requirement of helper compounds, predominant endosomal delivery, and/or are restricted to specific molecule classes. Photonic cell manipulation bears the potential to overcome these drawbacks. Here we investigated mammalian cell manipulation by single sub-nanosecond laser pulses. Axial beam waist positioning close to a cell monolayer induced culture vessel damage and zones of cell ablation. Cells at margins of ablation zones exhibited uptake of membrane-impermeant fluorophores and GFP expression plasmids. Increasing Rayleigh-length and beam waist diameter reduced the sensitivity to axial defocusing and resulted in robust molecule transfer. Serial application of single pulses focused over a moving cell monolayer yielded quantitative molecule transfer to cells at rates up to 40%. Our results could be basic to spatially and temporally controlled single laser pulse-mediated marker-free high throughput cell manipulation.

Comparative ex vivo Investigations on the Cutting Quality of the CO2 Laser and the Diode Pumped Er:YAG Laser.

Objectives: A sufficient histological evaluation is a key pillar in oncological treatment, especially in situations of cancer of unknown primary. CO2 laser technology is used in clinical routine of soft tissue surgery because of its cutting quality and availability. Diode pumped solid state Er(bium):YAG laser systems promise a higher cutting efficiency and minor thermal damages. The aim of this study was to compare both laser systems with respect to their suitability for cutting soft tissue. Methods: A setup was realized which enables comparable experiments with the clinical CO2 laser (AcuPulse 40ST DUO, Lumenis) and the Er:YAG laser system (DPM 40, Pantec Biosolutions AG). Fresh mucosal samples of porcine tongues were used to determine the influence of laser power and sample velocity on cutting depth and thermal damage width for both lasers. In addition, for the Er:YAG laser, the influence of the pulse repetition rate was examined additionally. For analysis, images of histological sections were taken. Results: In all experiments, the Er:YAG laser shows a significantly higher cutting depth (P < 0.0001) and less thermal damage width (P < 0.0001) than the CO2 laser. For example, at an average power of 7.7 W and a sample velocity of 5 mm/s the Er:YAG laser shows a mean cutting depth of 1.1 mm compared to the CO2 laser with 500 µm. While the Er:YAG laser shows a mean thermal damage width of 70 µm compared to 120 µm. Furthermore, the Er:YAG enables the adjustment of the cutting depth and thermal damage width by varying the irradiation parameters. A decrease of the repetition rate leads to a reduction of thermal damage. For example, a repetition rate of 100 Hz results in a thermal damage width of 46 µm compared to 87 µm at 800 Hz at an average power of 7.7 W and a cutting velocity = 5 mm/s while a homogenous cutting quality can be achieved. Conclusions: In conclusion, the results of these ex vivo experiments demonstrate significant advantages of the diode pumped Er:YAG laser system for soft tissue ablation compared to the CO2 laser, in particular regarding cutting efficiency and thermal damage width.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs.

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU µl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.

Development and verification of a snapshot dental intraoral three-dimensional scanner based on chromatic confocal imaging.

We describe the development and verification of an optical, powder-free, intraoral scanner based on a chromatic confocal imaging system, which has been realized in a single-shot multifocal approach. The system is based on a combination of micro-optical and dispersion optical elements. The methodology of recording and analyzing the acquired data are discussed in detail. A proof of concept with the application in intraoral scanning is provided. According to the current findings, the measurement uncertainty, scan speed, and overall performance of the device can well compete with the state-of-the-art of commercially available intraoral scanners.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO2) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Er:YAG laser activation of sodium hypochlorite for root canal soft tissue dissolution.

BACKGROUND AND OBJECTIVE: The aim of this in vitro study was to investigate the effect of Er:YAG laser irradiation on the ability of sodium hypochlorite (NaOCl) to dissolve soft tissue during endodontic procedures. MATERIALS AND METHODS: Two acrylic glass plates, each containing a semi-canal, were bolted together to form a complete canal. This geometry permitted one semi-canal to be filled with fine liver sausage of bovine origin dyed by methylene blue and the other with NaOCl (4.00-4.99% available chlorine; Sigma-Aldrich Corporation, St. Louis, MA), which was then activated by Er:YAG laser irradiation (KEY Laser 3; KaVo, Biberach, Germany) using a plain-ended fiber tip and a range of output energy and repetition rate. To achieve relatively low output energy from high input energy, the laser beam was attenuated by placing glass slides in the beam path. The resultant images acquired were analyzed using pixel-based analysis. Samples were statistically analyzed (two-way ANOVA, P < 0.05, univariate, bifactorial; IBM SPSS Statistics 19, SPSS Inc., Chicago, IL). RESULTS: Both output energy and repetition rate significantly influenced the tissue dissolution ability of NaOCl (P < 0.05). CONCLUSION: Within the limitations of this in vitro study, we conclude that laser activation of NaOCl at 200 mW output power leads to effective soft tissue dissolution. This finding can be of use to endodontists pursuing effective soft tissue dissolution from their irrigants.

Comparison of different focusing fiber tips for improved oral diode laser surgery.

BACKGROUND AND OBJECTIVES: State of the art for use of the fiber guided diode laser in dental therapy is the application of bare fibers. A novel concept with delivery fiber and exchangeable fiber tips enables the use of tips with special and optimized geometries for various applications. The aim of this study is the comparison of different focusing fiber tips for enhanced cutting efficacy in oral surgery. MATERIAL AND METHODS: For this purpose various designs of tip geometry were investigated and optimized by ray tracing simulations. Two applicators, one with a sphere, and another one with a taper, were realized and tested on porcine gingiva (diode laser, 940 nm, 5 W/cw; 7 W/modulated). The cutting depth and quality were determined by light microscope. Histological sections of the cuts were prepared by a cryo-microtome and microscopically analyzed to determine the cut depths and thermal damage zones. RESULTS: The simulations show that, using a sphere as fiber tip, an intensity increase of up to a factor of 16.2 in air, and 13.2 in water compared to a bare 200 µm fiber can be achieved. Although offering high focusing factor in water, the cutting quality of the sphere was rather poor. This is probably caused by a derogation of the focusing quality due to contamination during cutting and light scattering. Much better results were achieved with conically shaped fiber tips. Compared to bare fibers they exhibit improved handling properties with no hooking, more regular and deeper cuts (5 W/cw: 2,393 ± 468 µm, compared to the cleaved bare fiber 5 W/cw: 711 ± 268 µm). The thermal damage zones of the cuts are comparable for the various tips and fibers. CONCLUSIONS: In conclusion the results of our study show that cutting quality and efficiency of diode laser on soft tissue can be significantly improved using conically shaped fiber tips.

Thermal effects of white light illumination during microsurgery: clinical pilot study on the application safety of surgical microscopes.

Modern operating microscopes offer high power illumination to ensure optimal visualization, but can also cause thermal damage. The aim of our study is to quantify the thermal effects in vivo and discuss conditions for safe use. In a pilot study on volunteers, we measured the temperature at the skin surface during microscope illumination, including the influence of anaesthesia and the effects of staining, draping, or moistening of the skin. Irradiation within the limit given by safety regulations (200 mW/cm(2)) results in skin surface temperature of 43 degrees C. Higher intensities (forearm 335 mW/cm(2), back 250 mW/cm(2)) are tolerated, resulting in reversible hyperaemia. At a very high illumination intensity (750 mW/cm(2)), pain occurs within 30 s at temperatures of 46 degrees C+/-1 degrees C (hand and forearm), and 43 degrees C+/-2 degrees C (back), respectively. Anaesthesia has no distinct effect on the temperature, whereas staining and drapes result in much higher temperatures (>100 degrees C). Moistening at practicable flow rates can reduce temperature efficiently when combined with a light absorbing and water absorbent drape. In conclusion, surgeons must be aware that surgical microscope illumination without protective means can cause skin temperatures to rise much above pain threshold, which in our study serves as a (conservative) benchmark for potential damage.

Mechanism of high-power NIR laser bacteria inactivation.

Lasers are used in dentistry for a variety of indications. One of these is the disinfection of root canals or the sterilization of residual caries. Many studies have demonstrated the capacity to kill bacteria for lasers but the fundamental mechanism of the laser effect remains quite unclear. With our experiments we wanted to determine whether high-power NIR laser bacterial killing is caused by the light itself (photochemical effect) or by a photothermal process. In order to differentiate between mechanisms we heated bacteria suspensions of a nonpathogenic strain of E. coli by a water bath and by a diode laser (940 nm) with the same temporal temperature course. Furthermore, bacteria suspensions were irradiated while the temperature was fixed by ice water. Killing of bacteria was measured via fluorescence labelling. Comparison of killing rates between laser and water-based heating shows no significant differences. The most important parameter is the maximum temperature. Laser irradiation of bacteria at low temperatures does not result in killing. Our experiments show that at least for E. coli bacteria inactivation by high-power laser irradiation is solely based on a thermal process.

Fluorescence lifetime imaging (FLIM) of rhodamine 123 in living cells.

A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolving techniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits optical excitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitation light is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7 endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine 123(R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria, whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly, fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of the R123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium should not be above 25 microM.