Results of First In Vivo Trial of an Acoustic Self-Tonometer.

Purpose: Glaucoma is the world's most common cause of irreversible blindness, which makes early diagnosis, with the goal of preserving vision, essential. The current medical intervention is to reduce intraocular pressure (IOP) to slow down progression of the disease. The main goal of this study was to test a novel handheld acoustic self-tonometer on humans. Methods: A sound pressure pulse generated by a loudspeaker causes the eye to vibrate. A pressure chamber is placed on the human orbit to form a coupled system comprised of the patient's eye, the enclosed air, and the loudspeaker. A displacement sensor in front of the loudspeaker membrane allows the dynamic behavior of the entire system to be detected. Results: For this clinical trial series, a prototype of the acoustic self-tonometer principle was applied. The resulting membrane oscillation data showed sensitivity of patient IOP, but direct allocation of the measured damping and frequency to the IOP was not significant. For this reason, an artificial neural network was used to find relationships among the subjects' biometric eye parameters in combination with the self-tonometer data for the IOP reference. An expanded measurement uncertainty (kp = 2) equal to 6.53 mm Hg was determined for the self-tonometer in a Bland-Altman analysis using Goldmann applanation tonometer reference measurements. Conclusions: The usability and success rate of producing valid measurement values with the device during self-measurements by test subjects was nearly 92%. The cross-sensitivities observed require compensation in a possible redesign phase to reduce the measurement uncertainty by at least 25% to the maximum of 5 mm Hg required to seek medical device approval. Translational Relevance: Building on successful laboratory experiments with pig eyes, this article reports the results of testing the acoustic tonometer on humans.

Miniaturized Sample Preparation for Transmission Electron Microscopy.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible. A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM. The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics".

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.

Interaction between topographic/tomographic parameters and dry eye disease in keratoconus patients.

PURPOSE: To determine the interaction between corneal topographic and tomographic parameters and dry eye syndrome (DES) in keratoconus (KC) patients. METHODS: Seventy-seven eyes of 49 patients with KC (age 34.4 ± 11.6 years) were enrolled in this study. In these 77 eyes we recorded surface regularity index (SRI), surface asymmetry index (SAI) and Klyce/Maeda KC index (KCI) using the Topographic Modeling System (TMS-5, Tomey, Tennenlohe, Germany), Index of Surface Variance (ISV), Index of Vertical Asymmetry (IVA), KC Index (KI), Center KC Index (CKI), Index of Height Asymmetry (IHA) and Index of Height Decentration (IHD) using Pentacam (Pentacam HR, Oculus, Germany). Patients were subdivided into mild (grade 1-2) and severe stage (grade 3-4) KC groups according to Pentacam grading. To analyse tear film parameters we assessed in 77 KC eyes McMonnies questionnaire, Schirmer test and break-up time and in 26 eyes (10 eyes with mild KC, 16 eyes with severe KC) high-speed videokeratoscopy (during interblinking interval) using a novel commercially not available system (Tear Inspect). With Tear Inspect the analysed tear film parameters were (1) time of first irregularities of Placido rings and (2) time of eyelid closure. Patients were also subdivided into McMonnies questionnaire positive and negative groups. RESULTS: We did not find significant difference between patients with mild and severe KC in any of the examined tear film parameters (p > 0.66). There was no significant difference in SRI, SAI, KCI, ISV, IVA, KI, CKI, IHA and IHD in McMonnies test positive and negative KC patients (p > 0.07). There was no correlation between SRI, SAI, KCI, ISV, IVA, KI, CKI, IHA and IHD and any of the examined tear film parameters (without high-speed videokeratoscopy) neither in 77 KC patients nor in mild or severe KC eyes (r < 0.3). CONCLUSIONS: There is no interaction between DES and topographic/tomographic changes in KC-patients.

Single-cell lysis for visual analysis by electron microscopy.

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.

Connecting µ-fluidics to electron microscopy.

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.

Simultaneous examination of tear film break-up and the lipid layer of the human eye: a novel model eye for time course simulation of physiologic tear film behavior (Part 2).

This article describes a model eye created to mimic the three-layered human tear-film. The model replicates the properties of the mucin, liquid and lipid layer as well as the time-course behavior including blinking process and tear film break-up for measurements with the tear film sensor described in Part 1. The setup basically consists of a sphere that is moistened by a dilution of tear film substitute. Therefore, the time point of the first break-up strongly depends on the consistency of the dilution. By means of an integrated heater, the evaporation rate of the artificial tear-film can be increased. To illustrate the applicability of the model eye, exemplary images during tear-film simulation, taken with and without using the heater, are shown. All images were captured by the sensor. Results and other potential applications are briefly discussed.

Simultaneous examination of tear film break-up and the lipid layer of the human eye: a novel sensor design (Part 1).

This article presents a non-invasive sensor setup for objective analysis of the pre-corneal human tear-film involving a time-resolved videokeratoscopy and simultaneous imaging of the lipid layer of the tear-film by a second CMOS-camera. This paper describes in detail the mechanical and optical design of the sensor setup, the realization of the entire illumination component with an integrated Placido grid projection, and the calculation of the image formation using the Placido grid. This concept is demonstrated here with a test subject in the full inter-blink period. All images were taken under physiological conditions. The sensor can assist the ophthalmologist in diagnosing Dry-Eye Syndrome. Results and other potential applications are discussed.