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.

Cell adherence and drug delivery from particle based mesoporous silica films.

Spatially and temporally controlled drug delivery is important for implant and tissue engineering applications, as the efficacy and bioavailability of the drug can be enhanced, and can also allow for drugging stem cells at different stages of development. Long-term drug delivery over weeks to months is however difficult to achieve, and coating of 3D surfaces or creating patterned surfaces is a challenge using coating techniques like spin- and dip-coating. In this study, mesoporous films consisting of SBA-15 particles grown onto silicon wafers using wet processing were evaluated as a scaffold for drug delivery. Films with various particle sizes (100-900 nm) and hence thicknesses were grown onto trichloro(octadecyl)silane-functionalized silicon wafers using a direct growth method. Precise patterning of the areas for film growth could be obtained by local removal of the OTS functionalization through laser ablation. The films were incubated with the drug model 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), and murine myoblast cells (C2C12 cells) were seeded onto films with different particle sizes. Confocal laser scanning microscopy (CLSM) was used to study the cell growth, and a vinculin-mediated adherence of C2C12 cells on all films was verified. The successful loading of DiO into the films was confirmed by UV-vis and CLSM. It was observed that the drugs did not desorb from the particles during 24 hours in cell culture. During adherent growth on the films for 4 h, small amounts of DiO and separate particles were observed inside single cells. After 24 h, a larger number of particles and a strong DiO signal were recorded in the cells, indicating a particle mediated drug uptake. The vast majority of the DiO-loaded particles remained attached to the substrate also after 24 h of incubation, making the films attractive as longer-term reservoirs for drugs on e.g. medical implants.