On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Assessment of dynamic corneal nerve changes using static landmarks by in vivo large-area confocal microscopy-a longitudinal proof-of-concept study.

Background: The purpose of the present proof-of-concept study was to use large-area in vivo confocal laser scanning microscopy (CLSM) mosaics to determine the migration rates of nerve branching points in the human corneal subbasal nerve plexus (SNP). Methods: Three healthy individuals were examined roughly weekly over a total period of six weeks by large-area in vivo confocal microscopy of the central cornea. An in-house developed prototype system for guided eye movement with an acquisition time of 40 s was used to image and generate large-area mosaics of the SNP. Kobayashi-structures and nerve entry points (EPs) were used as fixed structures to enable precise mosaic registration over time. The migration rate of 10 prominent nerve fiber branching points per participant was tracked and quantified over the longitudinal period. Results: Total investigation times of 10 minutes maximum per participant were used to generate mosaic images with an average size of 3.61 mm2 (range: 3.18-4.42 mm2). Overall mean branching point migration rates of (46.4±14.3), (48.8±15.5), and (50.9±13.9) µm/week were found for the three participants with no statistically significant difference. Longitudinal analyses of nerve branching point migration over time revealed significant time-dependent changes in migration rate only in participant 3 between the last two measurements [(63.7±12.3) and (43.0±12.5) µm/week, P<0.01]. Considering individual branching point dynamics, significant differences in nerve migration rate from the mean were only found in a few exceptions. Conclusions: The results of this proof-of-concept study have demonstrated the feasibility of using in vivo confocal microscopy to study the migration rates of corneal subbasal nerves within large areas of the central human cornea (>1 mm2). The ability to monitor dynamic changes in the SNP opens a window to future studies of corneal nerve health and regenerative capacity in a number of systemic and ocular diseases. Since corneal nerves are considered part of the peripheral nervous system, this technique could also offer an objective diagnostic tool and biomarker for disease- or treatment-induced neuropathic changes.

In Vivo Monitoring of Corneal Dendritic Cells in the Subbasal Nerve Plexus during Trastuzumab and Paclitaxel Breast Cancer Therapy-A One-Year Follow-Up.

Paclitaxel and trastuzumab have been associated with adverse effects including chemotherapy-induced peripheral neuropathy (CIPN) or ocular complications. In vivo confocal laser scanning microscopy (CLSM) of the cornea could be suitable for assessing side effects since the cornea is susceptible to, i.e., neurotoxic stimuli. The study represents a one-year follow-up of a breast cancer patient including large-area in vivo CLSM of the subbasal nerve plexus (SNP), nerve function testing, and questionnaires during paclitaxel and trastuzumab therapy. Six monitoring sessions (one baseline, four during, and one after therapy) over 58 weeks were carried out. Large-area mosaics of the SNP were generated, and identical regions within all sessions were assigned. While corneal nerve morphology did not cause alterations, the number of dendritic cells (DCs) showed dynamic changes with a local burst at 11 weeks after baseline. Simultaneously, paclitaxel treatment was terminated due to side effects, which, together with DCs, returned to normal levels as the therapy progressed. Longitudinal in vivo CLSM of the SNP could complement routine examinations and be helpful to generate a comprehensive clinical picture. The applied techniques, with corneal structures acting as biomarkers could represent a diagnostic tool for the objective assessment of the severity of adverse events and the outcome.

Nonlinear interaction of Rayleigh waves in isotropic materials: Numerical and experimental investigation.

Surface treatment intensity monitoring is still an open and challenging nondestructive testing problem. For the estimation of residual stress with ultrasonic measurements, local linear and nonlinear elastic constants are needed as input. In this paper, nonlinear elastic-wave interactions (also called wave mixing or scattering) - namely, the generation of secondary ultrasonic waves in a nonlinear medium - are considered as a prospective means for near-surface nonlinear elastic parameter evaluation. The allowed interactions between bulk and surface waves, as well as the dependence of the scattering efficiency on the frequency and angle between source waves, were investigated through an analytical model, then compared with FEM simulations and experimental results. Finally, possible future steps for the development of the applied methods for the determination of near-surface higher-order elastic constants are discussed. In addition, several problem-relevant data processing procedures are presented.

Real-time large-area imaging of the corneal subbasal nerve plexus.

The morphometric assessment of the corneal subbasal nerve plexus (SNP) by confocal microscopy holds great potential as a sensitive biomarker for various ocular and systemic conditions and diseases. Automated wide-field montages (or large-area mosaic images) of the SNP provide an opportunity to overcome the limited field of view of the available imaging systems without the need for manual, subjective image selection for morphometric characterization. However, current wide-field montaging solutions usually calculate the mosaic image after the examination session, without a reliable means for the clinician to predict or estimate the resulting mosaic image quality during the examination. This contribution describes a novel approach for a real-time creation and visualization of a mosaic image of the SNP that facilitates an informed evaluation of the quality of the acquired image data immediately at the time of recording. In cases of insufficient data quality, the examination can be aborted and repeated immediately, while the patient is still at the microscope. Online mosaicking also offers the chance to identify an overlap of the imaged tissue region with previous SNP mosaic images, which can be particularly advantageous for follow-up examinations.

Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay.

Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.

Electromechanical Reciprocity Applied to the Sensing Properties of Guided Elastic Wave Transducers.

Guided elastic wave (GEW) transducers for structural health monitoring (SHM) can act as transmitters (senders) and receivers (sensors). Their performance in both cases depends on the structure to which they are coupled. Therefore, they must be characterized as system transducer- structure. The characterization of the transducer-structure as transmitter using a Scanning Laser Doppler Vibrometer (SLDV) is straightforward, whereas its characterization as receiver is non-trivial. We propose to exploit electromechanical reciprocity, which is an identity between the transfer functions of electrical-to-mechanical and mechanical-to-electrical conversions. For this purpose, the well-known electromechanical reciprocity theorem was adapted to the following situation: The two reciprocal states are "electrical excitation and detection of the surface velocity at point P" and "mechanical excitation at P and measurement of the electrical quantities". According to the derived formulas, the quantities on the mechanical and electrical sides must be chosen appropriately to ensure reciprocity as well as that the corresponding transfer functions are equal. We demonstrate the reciprocity with experimental data for correctly chosen transfer functions and show the deviation in reciprocity for a different choice. Furthermore, we propose further applications of electromechanical reciprocity.

Wide-field mosaics of the corneal subbasal nerve plexus in Parkinson's disease using in vivo confocal microscopy.

In vivo confocal microscopy (IVCM) is a non-invasive imaging technique facilitating real-time acquisition of images from the live cornea and its layers with high resolution (1-2 µm) and high magnification (600 to 800-fold). IVCM is extensively used to examine the cornea at a cellular level, including the subbasal nerve plexus (SBNP). IVCM of the cornea has thus gained intense interest for probing ophthalmic and systemic diseases affecting peripheral nerves. One of the main drawbacks, however, is the small field of view of IVCM, preventing an overview of SBNP architecture and necessitating subjective image sampling of small areas of the SBNP for analysis. Here, we provide a high-quality dataset of the corneal SBNP reconstructed by automated mosaicking, with an average mosaic image size corresponding to 48 individual IVCM fields of view. The mosaic dataset represents a group of 42 individuals with Parkinson's disease (PD) with and without concurrent restless leg syndrome. Additionally, mosaics from a control group (n = 13) without PD are also provided, along with clinical data for all included participants.

Burst of Corneal Dendritic Cells during Trastuzumab and Paclitaxel Treatment.

During breast cancer therapy, paclitaxel and trastuzumab are both associated with adverse effects such as chemotherapy-induced peripheral neuropathy and other systemic side effects including ocular complications. Corneal nerves are considered part of the peripheral nervous system and can be imaged non-invasively by confocal laser scanning microscopy (CLSM) on the cellular level. Thus, in vivo CLSM imaging of structures of the corneal subbasal nerve plexus (SNP) such as sensory nerves or dendritic cells (DCs) can be a powerful tool for the assessment of corneal complications during cancer treatment. During the present study, the SNP of a breast cancer patient was analyzed over time by using large-scale in vivo CLSM in the course of paclitaxel and trastuzumab therapy. The same corneal regions could be re-identified over time. While the subbasal nerve morphology did not alter significantly, a change in dendritic cell density and an additional local burst within the first 11 weeks of therapy was detected, indicating treatment-mediated corneal inflammatory processes. Ocular structures such as nerves and dendritic cells could represent useful biomarkers for the assessment of ocular adverse effects during cancer therapy and their management, leading to a better visual prognosis.

Morphological characterization of the human corneal epithelium by in vivo confocal laser scanning microscopy.

BACKGROUND: Regarding the growing interest and importance of understanding the cellular changes of the cornea in diseases, a quantitative cellular characterization of the epithelium is becoming increasingly important. Towards this, the latest research offers considerable improvements in imaging of the cornea by confocal laser scanning microscopy (CLSM). This study presents a pipeline to generate normative morphological data of epithelial cell layers of healthy human corneas. METHODS: 3D in vivo CLSM was performed on the eyes of volunteers (n=25) with a Heidelberg Retina Tomograph II equipped with an in-house modified version of the Rostock Cornea Module implementing two dedicated piezo actuators and a concave contact cap. Image data were acquired with nearly isotropic voxel resolution. After image registration, stacks of en-face sections were used to generate full-thickness volume data sets of the epithelium. Beyond that, an image analysis algorithm quantified en-face sections of epithelial cells regarding the depth-dependent mean of cell density, area, diameter, aggregation (Clark and Evans index of aggregation), neighbor count and polygonality. RESULTS: Imaging and cell segmentation were successfully performed in all subjects. Thereby intermediated cells were efficiently recognized by the segmentation algorithm while efficiency for superficial and basal cells was reduced. Morphological parameters showed an increased mean cell density, decreased mean cell area and mean diameter from anterior to posterior (5,197.02 to 8,190.39 cells/mm2; 160.51 to 90.29 µm2; 15.9 to 12.3 µm respectively). Aggregation gradually increased from anterior to posterior ranging from 1.45 to 1.53. Average neighbor count increased from 5.50 to a maximum of 5.66 followed by a gradual decrease to 5.45 within the normalized depth from anterior to posterior. Polygonality gradually decreased ranging from 4.93 to 4.64 sides of cells. The neighbor count and polygonality parameters exhibited profound depth-dependent changes. CONCLUSIONS: This in vivo study demonstrates the successful implementation of a CLSM-based imaging pipeline for cellular characterization of the human corneal epithelium. The dedicated hardware in combination with an adapted image registration method to correct the remaining motion-induced image distortions followed by a dedicated algorithm to calculate characteristic quantities of different epithelial cell layers enabled the generation of normative data. Further significant effort is necessary to improve the algorithm for superficial and basal cell segmentation.

How does grazing incidence ultrasonic microscopy work? A study based on grain-scale numerical simulations.

Grazing incidence ultrasonic microscopy (GIUM) is an experimental method for visualising the microstructures of polycrystals with local preferential orientations. It has previously been demonstrated on an austenitic stainless steel weld, exposing grains much smaller than the propagating wavelength, but the physical mechanism of the method has only been proposed as a hypothesis. In this paper, we use grain-scale finite element simulations based on the EBSD measurements to verify the principles behind GIUM images further and to assess how deep does the method penetrate the component under examination. The simulations indicate that while lateral contraction of grains contains microstructure signatures, the free surface effect is the crucial factor contributing to the generation of the images. Further, we show that only features up to the depth in the order of the average grain size in that direction can be visualised.

Parkinson's disease with restless legs syndrome-an in vivo corneal confocal microscopy study.

Small fiber neuropathy (SFN) has been suggested as a trigger of restless legs syndrome (RLS). An increased prevalence of peripheral neuropathy has been demonstrated in Parkinson's disease (PD). We aimed to investigate, in a cross-sectional manner, whether SFN is overrepresented in PD patients with concurrent RLS relative to PD patients without RLS, using in vivo corneal confocal microscopy (IVCCM) and quantitative sensory testing (QST) as part of small fiber assessment. Study participants comprised of age- and sex-matched PD patients with (n = 21) and without RLS (n = 21), and controls (n = 13). Diagnosis of RLS was consolidated with the sensory suggested immobilization test. Assessments included nerve conduction studies (NCS), Utah Early Neuropathy Scale (UENS), QST, and IVCCM, with automated determination of corneal nerve fiber length (CNFL) and branch density (CNBD) from wide-area mosaics of the subbasal nerve plexus. Plasma neurofilament light (p-NfL) was determined as a measure of axonal degeneration. No significant differences were found between groups when comparing CNFL (p = 0.81), CNBD (p = 0.92), NCS (p = 0.82), and QST (minimum p = 0.54). UENS scores, however, differed significantly (p = 0.001), with post-hoc pairwise testing revealing higher scores in both PD groups relative to controls (p = 0.018 and p = 0.001). Analysis of all PD patients (n = 42) revealed a correlation between the duration of L-dopa therapy and CNBD (ρ = -0.36, p = 0.022), and p-NfL correlated with UENS (ρ = 0.35, p = 0.026) and NCS (ρ = -0.51, p = 0.001). Small and large fiber neuropathy do not appear to be associated with RLS in PD. Whether peripheral small and/or large fiber pathology associates with central neurodegeneration in PD merits further longitudinal studies.

In vivo Histology of the Cornea - from the "Rostock Cornea Module" to the "Rostock Electronic Slit Lamp" - a Clinical "Proof of Concept" Study. / In-vivo-Histologie der Hornhaut ­ vom "Rostocker Cornea Modul" zur "Rostocker Elektronischen Spaltlampe" ­ eine klinische "Proof-of-Concept"-Studie.

INTRODUCTION: Confocal in vivo microscopy is an established method in ophthalmology research. As it requires contact coupling and calibration of the instruments is suboptimal, this method has been only rarely used in clinical routine work. As a result of close collaboration between physicists, information scientists and ophthalmologists, confocal laser scanning microscopy (CLSM) of the eye has been developed in recent years and a prototype can now be used in patients. The present study evaluates possible clinical uses of this method. MATERIAL AND METHODS: The essential innovations in CLSM are (1) a newly designed coupling element with superficial adaptation to corneal curvature and (2) the use of a dual computerised piezo drive for rapid and precise focusing. In post-processing and after elastic imaging registration of the individual images parallel to the surface, it is also possible to produce sagittal sections resembling a split lamp and with resolution in the micrometer range. The concept was tested on enucleated pig bulbi and tested on normal volunteers and selected patients with diseases of the cornea. RESULTS: Simultaneous imaging in planes parallel to the surface and in sagittal planes provided additional information that can help us to understand the processes of wound healing in all substructures of the cornea and the role of immune competent cells. Possible clinical uses were demonstrated in a volunteer with healthy eyes and several groups of patients (keratoconus after CXL, recurrent keratitis, status after PRK). These show that this new approach can be used in morphological studies at cellular level in any desired and appropriate test plane. CONCLUSIONS: It could be shown that this new concept of CLSM can be used clinically. It can provide valuable and novel information to both preclinical researchers and to ophthalmologists interested in corneal disease, e.g. density of Langerhans cells and epithelial stratification in ocular surface diseases.

A computerized spatial orientation test.

In three experiments, we compared performance on a paper-based perspective-taking task (the Spatial Orientation Test [SOT]; Hegarty & Waller, 2004) with performance on a computer-based version of the task. The computer-based version automates scoring angular errors, allows for different stimulus orders to be given to each participant, and allows for different testing time limits. In Experiment 1, the two media used different objects and mirror-image stimulus arrays in the two versions to mitigate the effects of memory for specific objects or responses. In Experiments 2 and 3, the two media used identical objects (also in a mirrored arrangement), to provide a more equivalent between-media comparison. We also substituted new objects for objects in the original version that had an inherent front/back (e.g., a car) and/or that were animate; directional or animate objects may add variance that is unrelated to perspective-taking ability. Experiment 3 used clarified instructions and a sample size sufficient to examine relatively small differences between the media as well as sex differences. Overall, the computer-based version produced performance that was similar to that of the paper-based version in terms of the rank-order of the participants. The new computer and paper versions of the SOT also had similar correlations with the Money Road Map test and the Santa Barbara Sense of Direction questionnaire, adding support to the claim that the computerized SOT is tapping into the same skill as the paper-based version. We provide a Java version of the new SOT, along with pdf files of instructions and practice stimuli, on the Open Science Framework website.

Improved SH0 Guided Wave Transducers Based on Piezoelectric Fiber Patches.

A piezoelectric fiber patch (PFP) is a transducer type that is suitable for guided-wave-based structural health monitoring (SHM) due to its light, thin, and flexible characteristics. In our previous work, a PFP-based transducer design for selective excitation of the zero-order shear horizontal wave mode (SH0) was introduced (shear horizontal PFP (SHPFP)). In this work, two modified SH0 wave PFP transducer designs are proposed: the rounded corner design and the dual design. The degree of improvement is determined by a numerical simulation and the dual design-the design with the most promise-is experimentally realized. Laser Vibrometry measured the generated wave field, confirming the results from the simulation. The new designs can generate an almost pure SH0 wave. The dual design has a very strong directivity that is useful for several guided-wave-based SHM applications. The conclusions on the design's properties as a transmitter are also valid for its properties as a sensor due to the reciprocity of piezoelectric transducers.

Corneal Subbasal Nerve Plexus Changes in Severe Diabetic Charcot Foot Deformity: A Pilot Study in Search for a DNOAP Biomarker.

INTRODUCTION: Diabetic neuroosteoarthropathy (DNOAP) early symptoms are unspecific, mimicking general infectious symptoms and rendering a diagnosis challenging. Consequently, unfavourable outcomes occur frequently, with recurrent foot ulceration, infectious complications, and eventually amputation. Corneal confocal microscopy (CCM) of the subbasal nerve plexus (SNP) is used to detect early peripheral neuropathy in diabetic patients without diabetic retinopathy. This pilot study was designed to determine if specific SNP changes manifest in severe DNOAP in comparison to a healthy control group. METHODS: This pilot study utilized a matched-pair analysis to investigate SNP changes by in vivo CCM for 26 patients (mean patient age 63.7 years, range 27 to 78) with severe DNOAP defined by condition after the need for reconstructive foot surgery (n = 13) and a healthy control group (n = 13). Corneal nerve fibre length (CNFL), nerve fibre density (CNFD), nerve branch density (CNBD), average weighted corneal nerve fibre thickness (CNFTh), nerve connecting points (CNCP), and average weighted corneal nerve fibre tortuosity (CNFTo) were assessed as well as the general clinical status, diabetic status, and ophthalmologic basic criteria. RESULTS: In vivo CCM revealed significantly reduced SNP parameters in the DNOAP group for CNFL (p = 0.010), CNFD (p = 0.037), CNBD (p = 0.049), and CNCP (p = 0.012) when compared to the healthy control group. Six patients (46%) of the DNOAP group suffered from diabetic retinopathy and none of the control group. CONCLUSIONS: This pilot study revealed a rarefication of SNP in all measured parameters in patients with severe DNOAP. We see a potential value of CCM providing a SNP-based biomarker for early stages of DNOAP prior to the development of any foot deformities that needs to be evaluated in further studies. This trial is registered with German Clinical Trials Register (DKRS) DRKS00007537.

Cellular in vivo 3D imaging of the cornea by confocal laser scanning microscopy.

We present an in vivo confocal laser scanning microscopy based method for large 3D reconstruction of the cornea on a cellular level with cropped volume sizes up to 266 x 286 x 396 µm3. The microscope objective used is equipped with a piezo actuator for automated, fast and precise closed-loop focal plane control. Furthermore, we present a novel concave surface contact cap, which significantly reduces eye movements by up to 87%, hence increasing the overlapping image area of the whole stack. This increases the cuboid volume of the generated 3D reconstruction significantly. The possibility to generate oblique sections using isotropic volume stacks opens the window to slit lamp microscopy on a cellular level.

Nonlinear effects of micro-cracks on long-wavelength symmetric Lamb waves.

For an elastic medium containing a homogeneous distribution of micro-cracks, an effective one-dimensional stress-strain relation has been determined with finite element simulations. In addition to flat micro-cracks, voids were considered that contain a Hertzian contact, which represents an example for micro-cracks with internal structure. The orientation of both types of micro-cracks was fully aligned or, for flat micro-cracks, totally random. For micro-cracks with Hertzian contacts, the case of random orientation was treated in an approximate way. The two types of defects were found to give rise to different degrees of non-analytic behavior of the effective stress-strain relation, which governs the nonlinear propagation of symmetric (S0) Lamb waves in the long-wavelength limit. The presence of flat micro-cracks causes even harmonics to grow linearly with propagation distance with amplitudes proportional to the amplitude of the fundamental wave, and gives rise to a static strain. The presence of the second type of defects leads to a linear growth of all harmonics with amplitudes proportional to the power 3/2 of the fundamental amplitude, and to a strain-dependent velocity shift. Simple expressions are given for the growth rates of higher harmonics of S0 Lamb waves in terms of the parameters occurring in the effective stress-strain relation. They have partly been determined quantitatively with the help of the FEM results for different micro-crack concentrations.

Dynamic Proteome Alteration and Functional Modulation of Human Saliva Induced by Dietary Chemosensory Stimuli.

Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium l-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-ß-sanshool (numbing), followed by tryptic digestion, nano-HPLC-MS/MS, and label-free protein quantitation demonstrated a stimulus- and time-dependent influence of the dietary chemosensates on salivation and the salivary proteome composition. Gene ontology enrichment analysis showed evidence for stimulus-induced alterations of the saliva proteome to boot an efficient molecular defense network of the oral cavity, e.g., 6-gingerol increased salivary lactoperoxidase activity, catalyzing the oxidation of thiocyanate to produce the antimicrobial and antifungal hypothiocyanate, from 0.37 ± 0.02 to 0.91 ± 0.05 mU/mL 45 s after stimulation. In comparison, oral citric acid stimulation induced an increase of myeloperoxidase activity, catalyzing the chloride oxidation to generate antimicrobial hypochloride in saliva, from 0.24 ± 0.04 to 0.70 ± 0.1 mU/mL as well as an increase of salivary levels of lysozyme, exhibiting antimicrobial activity on Gram-positive bacteria, from 6.0-10 to 100-150 µg/mL. Finally, microbial growth experiments clearly demonstrated for the first time that the increase of the salivary lysozyme abundance upon oral citric acid stimulation translates into an enhanced biological function, that is an almost complete growth inhibition of the two lysozyme-sensitive Gram-positive bacteria tested.

3D confocal laser-scanning microscopy for large-area imaging of the corneal subbasal nerve plexus.

The capability of corneal confocal microscopy (CCM) to acquire high-resolution in vivo images of the densely innervated human cornea has gained considerable interest in using this non-invasive technique as an objective diagnostic tool for staging peripheral neuropathies. Morphological alterations of the corneal subbasal nerve plexus (SNP) assessed by CCM have been shown to correlate well with the progression of neuropathic diseases and even predict future-incident neuropathy. Since the field of view of single CCM images is insufficient for reliable characterisation of nerve morphology, several image mosaicking techniques have been developed to facilitate the assessment of the SNP in large-area visualisations. Due to the limited depth of field of confocal microscopy, these approaches are highly sensitive to small deviations of the focus plane from the SNP layer. Our contribution proposes a new automated solution, combining guided eye movements for rapid expansion of the acquired SNP area and axial focus plane oscillations to guarantee complete imaging of the SNP. We present results of a feasibility study using the proposed setup to evaluate different oscillation settings. By comparing different image selection approaches, we show that automatic tissue classification algorithms are essential to create high-quality mosaic images from the acquired 3D datasets.