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.

Femtosecond laser direct writing of metal microstructure in a stretchable poly(ethylene glycol) diacrylate (PEGDA) hydrogel.

The fabrication of three-dimensional (3D) metal microstructures in a synthetic polymer-based hydrogel is demonstrated by femtosecond laser-induced photoreduction. The linear-shaped silver structure of approximately 2 micrometers in diameter is fabricated inside a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel. The silver structure is observed and confirmed by scanning electron microscopy (SEM) and elemental analysis using energy-dispersive X-ray spectroscopy (EDX). Shrinking and swelling of the fabricated structure is also demonstrated experimentally, which shows the potential of the present method for realizing 3D flexible electronic and optical devices, as well as for fabricating highly integrated devices at submicron scales.

Correlating 3D morphology with molecular pathology: fibrotic remodelling in human lung biopsies.

Assessing alterations of the parenchymal architecture is essential in understanding fibrosing interstitial lung diseases. Here, we present a novel method to visualise fibrotic remodelling in human lungs and correlate morphological three-dimensional (3D) data with gene and protein expression in the very same sample. The key to our approach is a novel embedding resin that clears samples to full optical transparency and simultaneously allows 3D laser tomography and preparation of sections for histology, immunohistochemistry and RNA isolation. Correlating 3D laser tomography with molecular diagnostic techniques enables new insights into lung diseases. This approach has great potential to become an essential tool in pulmonary research.

Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling.

The present report evaluates the advantages of using the gold nanoparticle-mediated laser perforation (GNOME LP) technique as a computer-controlled cell optoperforation to introduce Lucifer yellow (LY) into cells in order to analyze the gap junction coupling in cell monolayers. To permeabilize GM-7373 endothelial cells grown in a 24 multiwell plate with GNOME LP, a laser beam of 88 µm in diameter was applied in the presence of gold nanoparticles and LY. After 10 min to allow dye uptake and diffusion through gap junctions, we observed a LY-positive cell band of 179 ± 8 µm width. The presence of the gap junction channel blocker carbenoxolone during the optoperforation reduced the LY-positive band to 95 ± 6 µm. Additionally, a forskolin-related enhancement of gap junction coupling, recently found using the scrape loading technique, was also observed using GNOME LP. Further, an automatic cell imaging and a subsequent semi-automatic quantification of the images using a java-based ImageJ-plugin were performed in a high-throughput sequence. Moreover, the GNOME LP was used on cells such as RBE4 rat brain endothelial cells, which cannot be mechanically scraped as well as on three-dimensionally cultivated cells, opening the possibility to implement the GNOME LP technique for analysis of gap junction coupling in tissues. We conclude that the GNOME LP technique allows a high-throughput automated analysis of gap junction coupling in cells. Moreover this non-invasive technique could be used on monolayers that do not support mechanical scraping as well as on cells in tissue allowing an in vivo/ex vivo analysis of gap junction coupling.

Numerical far field simulations with the fast Fourier transformation and Fourier space interpolation.

As more complicated microscope systems are engineered, the amount of effects taken into account rises steadily. In this context we experienced the need for a simulation approach, that will deliver the intensity distribution in space and time for scanning laser microscopes. To achieve this goal, the frequency space representation of microscope objectives was used and adapted to determine their solution of the electromagnetic wave equation. We describe the steps necessary to efficiently implement an approach to simulate multidimensional solutions of the wave equation. This includes the connection between the back focal plane and the Fourier space representation as well as a proper interpolation method for the latter. The error-potential of our least erroneous interpolation, the power of hann (POH) interpolation, is compared to other common interpolation methods. Finally we demonstrate the current potential of the approach by simulating an "expanding" optical vortex focus.

Single shot telecentricity measurement by Fourier space grid separation.

The experimental documentation of the properties of an optical system represents a particular challenge. Besides the measurement of focal quality and field distortions, telecentric systems have to yield a parallel beam propagation direction. In this paper we propose a method to test, document and optimize the telecentricity of a laser scanning system by scanning two crossed polka dot beam splitters at once. By separating both beam splitters in Fourier space we were able to detect tilting angles below 2 · 10(-3) rad for four different laser wavelengths within the same optical system. By this we determined the optimum system parameters for our scanning laser optical tomography (SLOT) setup.

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine.

BACKGROUND: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. RESULTS: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. CONCLUSION: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Non-invasive in vivo imaging by confocal laser scanning microscopy of gingival tissues following natural plaque deposition.

AIM: Imaging with Confocal Laser Scanning Microscopy (CLSM) generates high-resolution images and may be well suited for basic research in Periodontology and Implant Dentistry. The present study was aimed to explore the in vivo application of CLSM in experimentally induced gingivitis. MATERIALS AND METHODS: Ten subjects were recruited and were advised to stop any oral hygiene of the upper front teeth for 7 days. The gingival tissues were observed using a Heidelberg Retina Tomograph combined with a Rostock Cornea Module at baseline and day 7. The system used a laser of 670 nm and the contrast was given by backscattering from different tissues. Each examination created 800-1200 images that were descriptively analysed. RESULTS: After 7 days of abandoned oral hygiene, plaque scores and bleeding frequencies increased. By using CLSM images tooth hard substances, cells and plaque deposits were distinguishable. Increased epithelial cell irregularities, the apical migration of the sulcular epithelium, cellular infiltrates within the sulcus and plaque deposits were observed at day 7. CONCLUSIONS: The present study showed for the first time that CLSM is suitable for in vivo imaging of the gingival sulcus and adjacent tissues.

Imperfect refractive index matching in scanning laser optical tomography and a method for digital correction.

Significance: Scanning laser optical tomography (SLOT) is a volumetric multi-modal imaging technique that is comparable to optical projection tomography and computer tomography. Image quality is crucially dependent on matching the refractive indexes (RIs) of the sample and surrounding medium, but RI matching often requires some effort and is never perfect. Aim: Reducing the burden of RI matching between the immersion medium and sample in biomedical imaging is a challenging and interesting task. We aim at implementing a post processing strategy for correcting SLOT measurements that have errors caused by RI mismatch. Approach: To better understand the problems with poorly matched Ris, simulated SLOT measurements with imperfect RI matching of the sample and medium are performed and presented here. A method to correct distorted measurements was developed and is presented and evaluated. This method is then applied to a sample containing fluorescent polystyrene beads and a sample made of olydimethylsiloxane with embedded fluorescent nanoparticles. Results: From the simulations, it is evident that measurements with an RI mismatch larger than 0.02 and no correction yield considerably worse results compared to perfectly matched measurements. RI mismatches larger than 0.05 make it almost impossible to resolve finer details and structures. By contrast, the simulations imply that a measurement with an RI mismatch of up to 0.1 can still yield reasonable results if the presented correction method is applied. The experiments validate the simulated results for an RI mismatch of about 0.09. Conclusions: The method significantly improves the SLOT image quality for samples with imperfectly matched Ris. Although the absolutely best imaging quality will be achieved with perfect RI matching, these results pave the way for imaging in SLOT with RI mismatches while maintaining high image quality.

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.

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.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Femtosecond laser induced step-like structures inside transparent hydrogel due to laser induced threshold reduction.

In the area of laser material processing, versatile applications for cutting glasses and transparent polymers exist. However, parasitic effects such as the creation of step-like structures appear when laser cutting inside a transparent material. To date, these structures were only described empirically. This work establishes the physical and chemical mechanisms behind the observed effects and describes the influence of process and material parameters onto the creation of step-like structures in hydrogel, Dihydroxyethylmethacrylat (HEMA). By focusing laser pulses in HEMA, reduced pulse separation distance below 50 nm and rise in pulse energy enhances the creation of unintended step-like structures. Spatial resolved Raman-spectroscopy was used to measure the laser induced chemical modification, which results into a reduced breakdown threshold. The reduction in threshold influences the position of optical breakdown for the succeeding laser pulses and consequently leads to the step-like structures. Additionally, the experimental findings were supplemented with numerical simulations of the influence of reduced damage threshold onto the position of optical breakdown. In summary, chemical material change was defined as cause of the step-like structures. Furthermore, the parameters to avoid these structures were identified.

Enabling second harmonic generation as a contrast mechanism for optical projection tomography (OPT) and scanning laser optical tomography (SLOT).

Volumetric imaging of connective tissue provides insights into the structure of biological tissue. Second harmonic generation (SHG) microscopy has become a standard method to image collagen rich tissue like skin or cornea. Due to the non-centrosymmetric architecture, no additional label is needed and tissue can be visualized noninvasively. Thus, SHG microscopy enables the investigation of collagen associated diseases, providing high resolution images and a field of view of several hundreds of µm. However, the in toto visualization of larger samples is limited to the working distance of the objective and the integration time of the microscope setup, which can sum up to several hours and days. A faster imaging technique for samples in the mesoscopic range is scanning laser optical tomography (SLOT), which provides linear fluorescence, scattering and absorption as intrinsic contrast mechanisms. Due to the advantages of SHG and the reduced measurement time of SLOT, the integration of SHG in SLOT would be a great extension. This way SHG measurements could be performed faster on large samples, providing isotropic resolution and simultaneous acquisition of all other contrast mechanisms available, such as fluorescence and absorption. SLOT is based on the principle of computed tomography, which requires the rotation of the sample. The SHG signal, however, depends strongly on the sample orientation and the polarization of the laser, which results in SHG intensity fluctuation during sample rotation and prevents successful 3D reconstruction. In this paper we investigate the angular dependence of the SHG signal by simulation and experiment and found a way to eliminate reconstruction artifacts caused by this angular dependence in SHG-SLOT data. This way, it is now possible to visualize samples in the mesoscopic range using SHG-SLOT, with isotropic resolution and in correlation to other contrast mechanisms as absorption, fluorescence and scattering.

Time resolved 3D live-cell imaging on implants.

It is estimated that two million new dental implants are inserted worldwide each year. Innovative implant materials are developed in order to minimize the risk of peri-implant inflammations. The broad range of material testing is conducted using standard 2D, terminal, and invasive methods. The methods that have been applied are not sufficient to monitor the whole implant surface and temporal progress. Therefore, we built a 3D peri-implant model using a cylindrical implant colonized by human gingival fibroblasts. In order to monitor the cell response over time, a non-toxic LIVE/DEAD staining was established and applied to the new 3D model. Our LIVE/DEAD staining method in combination with the time resolved 3D visualization using Scanning Laser Optical Tomography (SLOT), allowed us to monitor the cell death path along the implant in the 3D peri-implant model. The differentiation of living and dead gingival fibroblasts in response to toxicity was effectively supported by the LIVE/DEAD staining. Furthermore, it was possible to visualize the whole cell-colonized implant in 3D and up to 63 hours. This new methodology offers the opportunity to record the long-term cell response on external stress factors, along the dental implant and thus to evaluate the performance of novel materials/surfaces.

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.