Einsatz ultrakurzer Laserpulse in der refraktiven Laserchirugie

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Intracellular Cargo Delivery Induced by Irradiating Polymer Substrates with Nanosecond-Pulsed Lasers

There is a great need in the biomedical field to efficiently, and cost-effectively, deliver membrane-impermeable molecules into the cellular cytoplasm. However, the cell membrane is a selectively permeable barrier, and large molecules often cannot pass through the phospholipid bilayer. We show that nanosecond laser-activated polymer surfaces of commercial polyvinyl tape and black polystyrene Petri dishes can transiently permeabilize cells for high-throughput, diverse cargo delivery of sizes of up to 150 kDa. The polymer surfaces are biocompatible and support normal cell growth of adherent cells. We determine the optimal irradiation conditions for poration, influx of fluorescent molecules into the cell, and post-treatment viability of the cells. The simple and low-cost substrates we use have no thin-metal structures, do not require cleanroom fabrication, and provide spatial selectivity and scalability for biomedical applications

Superresolved femtosecond laser nanosurgery of cells

We report on femtosecond nanosurgery of fluorescently labeled structures in cells with a spatially superresolved laser beam. The focal spot width is reduced using phase filtering applied with a programmable phase modulator. A comprehensive statistical analysis of the resulting cuts demonstrates an achievable average resolution enhancement of 30 %

Spatially-targeted laser fabrication of multi-metal microstructures inside a hydrogel

The spatially-targeted fabrication of bimetallic microstructures coexisting in the supporting hydrogel is demonstrated by multi-photon photoreduction. Microstructures composed of gold and silver were fabricated along a predefined trajectory by taking advantages of the hydrogel's ionic permeability. Different resonant wavelengths of optical absorption were obtained for gold, silver, and their bimetallic structures. Transmission electron microscopy and energy dispersive X-ray analysis revealed that the optical properties are attributable to the formation of bimetallic structure consisted of core-shell nanoparticles. The fabrication of dissimilar metal structures within hydrogel is a promising technique for optically driven actuators in soft robotics and sensing applications by allowing for site-selective optical properties. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Parameters for Optoperforation-Induced Killing of Cancer Cells Using Gold Nanoparticles Functionalized With the C-terminal Fragment of Clostridium Perfringens Enterotoxin

Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10−11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells

3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation

3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation

3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation

3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation

Biodegradable microsphere-mediated cell perforation in microfluidic channel using femtosecond laser

The use of small particles has expanded the capability of ultrashort pulsed laser optoinjection technology toward simultaneous treatment of multiple cells. The microfluidic platform is one of the attractive systems that has obtained synergy with laser-based technology for cell manipulation, including optoinjection. We have demonstrated the delivery of molecules into suspended-flowing cells in a microfluidic channel by using biodegradable polymer microspheres and a near-infrared femtosecond laser pulse. The use of polylactic-co-glycolic acid microspheres realized not only a higher optoinjection ratio compared to that with polylactic acid microspheres but also avoids optical damage to the microfluidic chip, which is attributable to its higher optical intensity enhancement at the localized spot under a microsphere. Interestingly, optoinjection ratios to nucleus showed a difference for adhered cells and suspended cells. The use of biodegradable polymer microspheres provides high throughput optoinjection; i.e., multiple cells can be treated in a short time, which is promising for various applications in cell analysis, drug delivery, and ex vivo gene transfection to bone marrow cells and stem cells without concerns about residual microspheres. © 2016 Society of Photo-Optical Instrumentation Engineers (SPIE).JSPS KAKENHI for Challenging Exploratory Research/2656026

Signal and response properties indicate an optoacoustic effect underlying the intra-cochlear laser-optical stimulation

Optical cochlea stimulation is under investigation as a potential alternative to conventional electric cochlea implants in treatment of sensorineural hearing loss. If direct optical stimulation of spiral ganglion neurons (SGNs) would be feasible, a smaller stimulation volume and, therefore, an improved frequency resolution could be achieved. However, it is unclear whether the mechanism of optical stimulation is based on direct neuronal stimulation or on optoacoustics. Animal studies on hearing vs. deafened guinea pigs already identified the optoacoustic effect as potential mechanism for intra-cochlear optical stimulation. In order to characterize the optoacoustic stimulus more thoroughly the acoustic signal along the beam path of a pulsed laser in water was quantified and compared to the neuronal response properties of hearing guinea pigs stimulated with the same laser parameters. Two pulsed laser systems were used for analyzing the influence of variable pulse duration, pulse energy, pulse peak power and absorption coefficient. Preliminary results of the experiments in water and in vivo suggesta similar dependency of response signals on the applied laser parameters: Both datasets show an onset and offset signal at the beginning and the end of the laser pulse. Further, the resulting signal amplitude depends on the pulse peak power as well as the temporal development of the applied laser pulse. The data indicates the maximum of the first derivative of power as the decisive factor. In conclusion our findings strengthen the hypothesis of optoacoustics as the underlying mechanism for optical stimulation of the cochlea. © SPIE 201

Evaluation of laser induced sarcomere microdamage: Role of damage extent and location in cardiomyocytes

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laserbased nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6-7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms. Copyright