Acoustic stimulation of the human round window by laser-induced nonlinear optoacoustics.

The feasibility of low frequency pure tone generation in the inner ear by laser-induced nonlinear optoacoustic effect at the round window was demonstrated in three human cadaveric temporal bones (TB) using an integral pulse density modulation (IPDM). Nanosecond laser pulses with a wavelength in the near-infrared (NIR) region were delivered to the round window niche by an optical fiber with two spherical lenses glued to the end and a viscous gel at the site of the laser focus. Using IPDM, acoustic tones with frequencies between 20 Hz and 1 kHz were generated in the inner ear. The sound pressures in scala tympani and vestibuli were recorded and the intracochlear pressure difference (ICPD) was used to calculate the equivalent sound pressure level (eq. dB SPL) as an equivalent for perceived loudness. The results demonstrate that the optoacoustic effect produced sound pressure levels ranging from 140 eq. dB SPL at low frequencies ≤ 200 Hz to 90 eq. dB SPL at 1 kHz. Therefore, the produced sound pressure level is potentially sufficient for patients requiring acoustic low frequency stimulation. Hence, the presented method offers a potentially viable solution in the future to provide the acoustic stimulus component in combined electro-acoustic stimulation with a cochlear implant.

Imaging and quantification of the tumor microenvironment of triple negative breast cancer using TPEF and scanning laser optical tomography.

Triple-negative breast cancer is an aggressive subtype of breast cancer that has a poor five-year survival rate. The tumor's extracellular matrix is a major compartment of its microenvironment and influences the proliferation, migration and the formation of metastases. The study of such dependencies requires methods to analyze the tumor matrix in its native form. In this work, the limits of SHG-microscopy, namely limited penetration depth, sample size and specificity, are addressed by correlative three-dimensional imaging. We present the combination of scanning laser optical tomography (SLOT) and multiphoton microscopy, to depict the matrix collagen on different scales. Both methods can be used complementarily to generate full-volume views and allow for in-depth analysis. Additionally, we explore the use of SHG as a contrast mechanism for complex samples in SLOT. It was possible to depict the overall collagen structure and specific fibers using marker free imaging on different scales. An appropriate sample preparation enables the fixation of the structures while simultaneously conserving the fluorescence of antibody staining. We find that SHG is a suitable contrast mechanism to depict matrix collagen even in complex samples and using SLOT. The insights presented here shall further facilitate the study of the tumor extracellular matrix by correlative 3d imaging.

Optoacoustic tones generated by nanosecond laser pulses can cover the entire human hearing range.

The aim of this work is to generate defined tones that cover the human hearing range in aqueous media for a later application in middle or inner ear implants. In our experiments, we investigated the characteristics of single laser pulses and pulse trains with different laser repetition rates of nanosecond laser pulses that were focused into aqueous media in a small volume. The frequency of the generated tones was limited by the spectral properties of the single acoustic pulses, which depended on the medium. Tones with fundamental frequencies above 8 kHz were generated using laser pulses focused into water. By replacing water with gel, tones between 500 Hz and 20 kHz could be produced. The generation of tones in the low-frequency range was only possible when laser pulse trains with pulse density modulated pulse patterns were applied in gel. This enabled the generation of tones between 20 Hz and 2 kHz. Consequently, the combination of different pulse patterns for the different frequency ranges allows generating optoacoustic tones between 20 Hz and 20 kHz in gel. Thus, we can cover the complete range of human hearing through optoacoustically generated tones.

Measurement of tear resistance after manual capsulorhexis and femtosecond laser-assisted capsulotomy of crystalline lenses.

BACKGROUND: In this study, the tear resistance of porcine lens capsules after continuous curvilinear capsulorhexis (CCC) and femtosecond (fs)-laser-assisted capsulotomy for cataract surgery (FLC) with different laser parameters is measured with a custom-made testing setup. METHODS: Forty-five fresh porcine lenses were randomly chosen for CCC (n = 15) or FLC 1 (n = 15) and FLC 2 (n = 15). The FLC 1-group was treated with smaller spot distances than the FLC 2-group. The force necessary to break the opening of the anterior capsule and the maximum displacement were measured. RESULTS: The mean tear resistance of the CCC-group (150 ± 70 mN) was higher than that of the FLC 1-group (60 ± 20 mN) and the FLC 2-group (30 ± 20 mN). CONCLUSION: It could be shown that CCC leads to a significantly higher tear resistance of the opening than FLC in porcine lenses. The femtosecond laser group demonstrated that smaller spot distances lead to a higher tear resistance.

Corneal riboflavin gradients and UV-absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1 to 0.5.

Avoiding damage of the endothelial cells, especially in thin corneas, remains a challenge in corneal collagen crosslinking (CXL). Knowledge of the riboflavin gradients and the UV absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1% to 0.5% could optimize the treatment. In this study, we present a model to calculate the UV-intensity depending on the corneal thickness. Ten groups of de-epithelialized porcine corneas were divided into 2 subgroups. Five groups received an imbibition of 10 min and the other five groups for 30 min. The applied riboflavin concentrations were 0.1%, 0.2%, 0.3%, 0.4% and 0.5% diluted in a 15% dextran solution for each subgroup. After the imbibition process, two-photon fluorescence microscopy was used to determine fluorescence intensity, which was compared to samples after saturation, yielding the absolute riboflavin concentration gradient of the cornea. The extinction coefficient of riboflavin solutions was measured using a spectrophotometer. Combining the obtained riboflavin concentrations and the extinction coefficients, a depth-dependent UV-intensity profile was calculated for each group. With increasing corneal depth, the riboflavin concentration decreased for all imbibition solutions and application times. The diffusion coefficients of 10 min imbibition time were higher than for 30 min. A higher RF concentration and a longer imbibition time resulted in higher UV-absorption and a lower UV-intensity in the depth of the cornea. Calculated UV-transmission was 6 percentage points lower compared to the measured transmission. By increasing the riboflavin concentration of the imbibition solution, a substantially higher UV-absorption inside the cornea is achieved. This offers a simple treatment option to control the depth of crosslinking e.g. in thin corneas, resulting in a lower risk of endothelial damage.

Light-cell interactions in depth-resolved optogenetics.

Light as a tool in medical therapy and biological research has been studied extensively and its application is subject to continuous improvement. However, safe and efficient application of light-based methods in photomedicine or optogenetics requires knowledge about the optical properties of the target tissue as well as the response characteristics of the stimulated cells. Here, we used tissue phantoms and a heart-like light-sensitive cell line to investigate optogenetic stimulation through tissue layers. The input power necessary for successful stimulation could be described as a function of phantom thickness. A model of light transmission through the tissue phantoms gives insights into the expected stimulation efficiency. Cell-type specific effects are identified that result in deviations of the stimulation threshold from the modelled predictions. This study provides insights into the complex interplay between light, tissue and cells during deep-tissue optogenetics. It can serve as an orientation for safe implementation of light-based methods in vivo.

PEGDMA Hydrogels for Cell Adhesion and Optical Waveguiding.

Hydrogels are favored materials in tissue engineering as they can be used to imitate tissues, provide scaffolds, and guide cell behavior. Recent advances in the field of optogenetics have created a need for biocompatible optical waveguides, and hydrogels have been investigated to meet these requirements. However, combining favorable waveguiding characteristics, high biocompatibility, and controllable bioactivity in a single device remains challenging. Here, we investigate the use of poly(ethylene glycol) hydrogels as carriers and illumination systems for in vitro cell culture. We present a comprehensive and reproducible protocol for selective bioactivation of the hydrogels, achieving high proliferation rates and strong cell adhesion on the treated surface. A cell model expressing the photoconvertible fluorescent protein Dendra2 confirmed that light-cell interactions occur at the hydrogel surface. Monte Carlo simulations were performed as a tool to predict the extent of these interactions. This study demonstrates a hydrogel-based waveguiding system for targeted cell stimulation in vitro and potentially in vivo environments.

Gold nanoparticle-mediated laser stimulation induces a complex stress response in neuronal cells.

Stimulation of neuronal cells generally resorts to electric signals. Recent advances in laser-based stimulation methods could present an alternative with superior spatiotemporal resolution. The avoidance of electronic crosstalk makes these methods attractive for in vivo therapeutic application. In particular, nano-mediators, such as gold nanoparticles, can be used to transfer the energy from a laser pulse to the cell membrane and subsequently activate excitable cells. Although the underlying mechanisms of neuronal activation have been widely unraveled, the overall effect on the targeted cell is not understood. Little is known about the physiological and pathophysiological impact of a laser pulse targeted onto nanoabsorbers on the cell membrane. Here, we analyzed the reaction of the neuronal murine cell line Neuro-2A and murine primary cortical neurons to gold nanoparticle mediated laser stimulation. Our study reveals a severe, complex and cell-type independent stress response after laser irradiation, emphasizing the need for a thorough assessment of this approach's efficacy and safety.