MAVIS: an integrated system for live microscopy and vitrification.

Cryo-electron microscopy of vitrified biological samples can provide three-dimensional reconstructions of macromolecules and organelles within bacteria and cells at nanometer scale resolution, even in native conditions. Localization of specific structures and imaging of cellular dynamics in cellular cryo-electron microscopy is limited by (i) the use of cryo-fixation to preserve cellular structures, (ii) the restricted availability of electron dense markers to label molecules inside cells and (iii) the inherent low contrast of cryo electron microscopy. These limitations can be mitigated to a large extend by correlative light and electron microscopy, where the sample is imaged by both light and electron microscopy. Here we present a Microscopy and Vitrification Integrated System (MAVIS) that combines a light microscope with a plunger to vitrify thin specimens. MAVIS provides the capability for fluorescence light microscopic imaging of living cells and bacteria that are adhered to an electron microscopy grid and subsequent vitrification within a time frame of seconds. The instrument allows targeting of dynamic biological events in time and space by fluorescence microscopy for subsequent cryo light and electron microscopy. Here we describe the design and performance of the MAVIS, illustrated with biological examples.

Standardized and automated solid-phase extraction procedures for high-throughput proteomics of body fluids.

In order to balance the speed of analytical sample preparation procedures with mass spectrometry (MS)-based clinical proteomics the application of high-throughput robotic systems for body fluid workup is essential. In this paper we describe the implementation of various solid-phase extraction (SPE) sample preparation protocols on two different platforms, namely: 1) Magnetic bead-based SPE of peptides and proteins from body fluids on a Hamilton liquid handling workstation; 2) Cartridge-based SPE on a SPARK Symbiosis system. All SPE protocols were optimized for MS-based proteomics and compared with respect to obtained peptide- and protein profiles. Throughput numbers that were achieved in a 24 hour time frame for the sample workup procedures were more than 700 samples for the magnetic bead-based method and over 1000 samples for the cartridge-based method.

Comparison of CMT1A and CMT2: similarities and differences.

To evaluate the clinical and electrophysiological similarities and differences between two large groups of patients with Charcot-Marie-Tooth disease, i.e. CMT1A and CMT2, we performed a post hoc comparison of clinical and electrophysiological data. Most CMT1A and CMT2 patients had the classical CMT phenotype. Age of onset was significantly later in CMT2. Total areflexia was present in approximately half of the CMT1A patients whereas it was rare in CMT2. Foot deformities and weakness of knee extensor and foot dorsal flexor muscles were more frequent in CMT1A. Median nerve motor nerve conduction velocities (MNCV) were always less than 38 m/s in CMT1A patients, whereas this was also the case in 16% of the CMT2 patients. Sensory nerve conduction velocities showed less overlap. In both CMT1A and CMT2 CMAP and SNAP amplitudes were often reduced or not obtainable in the legs. In CMT1A, SNAP amplitude was more reduced and SNAP duration more prolonged than in CMT2. We conclude that there are no robust clinical signs or symptoms that differentiate between CMT1A and CMT2 patients. Electrodiagnostical studies show a length-dependent motor and sensory axonal dysfunction in both CMT-types. Additional SNAP and SNCV evaluation may be helpful in focusing molecular genetic analysis in the occasional case of CMT2 showing slow motor nerve conduction velocities overlapping with CMT1A values. The reduction of CMAP and SNAP amplitudes in CMT1A is probably a combined effect of demyelination and axonal dysfunction.

Eyelid movements: behavioral studies of blinking in humans under different stimulus conditions.

The kinematics and neurophysiological aspects of eyelid movements were examined during spontaneous, voluntary, air puff, and electrically induced blinking in healthy human subjects, using the direct magnetic search coil technique simultaneously with electromyographic recording of the orbicularis oculi muscles (OO-EMG). For OO-EMG recordings, surface electrodes were attached to the lower eyelids. To measure the vertical lid displacement, a search coil with a diameter of 3 mm was placed 1 mm from the rim on the upper eyelid on a marked position. Blink registrations were performed from the zero position and from 28 randomly chosen positions. Blinks elicited by electrical stimulation of the supraorbital nerve had shortest duration and were least variable. In contrast, spontaneous blinks had longer duration and greater variability. Blinks induced by air puff had a slightly longer duration and similar variability as electrically induced blinks. There was a correlation between the maximal down phase amplitude and the integrated OO-EMG. Blink duration and maximal down phase amplitude were affected by eye position. Eyes positioned 30 degrees above horizontal displayed the shortest down phase duration and the largest maximal down phase amplitude and velocity. At 30 degrees below horizontal, blinks had the longest total duration, the longest down phase duration, and the lowest maximal down phase amplitude and velocity. The simultaneously recorded integrated OO-EMG was largest in the 30 degrees downward position. In four subjects, the average blinking data showed a linear relation between eye position and OO-EMG, maximal down phase amplitude, and maximal downward velocity.