[Super-resolution optical microscopy of the organ of Corti. Investigations on the fine structure of the inner hair cell afferent synapse by the 4Pi and STED techniques]. / Höchstauflösende optische Mikroskopie am Corti-Organ. Feinstrukturaufklärung an der afferenten Synapse innerer Haarzellen mittels 4Pi- und STED-Technik.

BACKGROUND: Inner hair cells encode sound into action potentials in the auditory nerve. Spiral ganglion neurons form the afferent innervation of inner hair cells via the hair cell synapse. The structure and function of this ribbon-type synapse is considered to have a major impact on the sound encoding process itself. In this study we have used conventional confocal microscopy as well as super-resolution techniques to investigate the synaptic organization in the inner hair cells of mice. MATERIAL AND METHODS: Functionally relevant proteins of the afferent inner hair cell synapse were selectively marked using immunohistochemical methods and investigated with conventional confocal and super-resolution 4Pi- and stimulated emission depletion (STED) techniques. RESULTS: Synapse and innervation density was mapped over the entire tonotopic axis. We found inner hair cells in the region of best hearing to have about twice the number of afferent fibres compared to the apex or base of the cochlea. For the first time 4Pi and STED microscopic techniques were employed to resolve the fine structure of these synapses beyond the resolution of conventional light microscopy. With 4Pi a resolution of approximately 100 nm in the z-axis direction is feasible. In practice STED delivers an effective resolution between 150 and 30 nm, depending on the power of the lasers employed. Synapses at different tonotopic positions of the cochlea exhibit no relevant structural differences at this level of resolution. The 4Pi and STED microscopic techniques are capable of showing the structure of afferent synapses in the organ of Corti with unsurpassed resolution. These images contribute to our understanding of sound-encoding mechanisms in the inner ear.

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment.

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross-talk between adjacent imaging channels. We demonstrate that a time-multiplexed non-linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single-photon confocal system. The background becomes irrelevant for thin (< 15 microm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.

Telehealth: reaching out to newly injured spinal cord patients.

OBJECTIVES: The authors present preliminary results on health-related outcomes of a randomized trial of telehealth interventions designed to reduce the incidence of secondary conditions among people with mobility impairment resulting from spinal cord injury (SCI). METHODS: Patients with spinal cord injuries were recruited during their initial stay at a rehabilitation facility in Atlanta. They received a video-based intervention for nine weeks, a telephone-based intervention for nine weeks, or standard follow-up care. Participants are followed for at least one year, to monitor days of hospitalization, depressive symptoms, and health-related quality of life. RESULTS: Health-related quality of life was measured using the Quality of Well-Being (QWB) scale. QWB scores (n = 111) did not differ significantly between the three intervention groups at the end of the intervention period. At year one post discharge, however, scores for those completing one year of enrollment (n = 47) were significantly higher for the intervention groups compared to standard care. Mean annual hospital days were 3.00 for the video group, 5.22 for the telephone group, and 7.95 for the standard care group. CONCLUSIONS: Preliminary evidence suggests that in-home telephone or video-based interventions do improve health-related outcomes for newly injured SCI patients. Telehealth interventions may be cost-saving if program costs are more than offset by a reduction in rehospitalization costs, but differential advantages of video-based interventions versus telephone alone warrant further examination.

Time-multiplexed multifocal multiphoton microscope.

We resolve the classical conflict between parallelization and axial resolution in three-dimensional fluorescence microscopy through time-multiplexed multifocal multiphoton excitation. A rotating array of microlenses on a disk splits ultrafast laser pulses in such a way that an array of high-aperture foci are created in the sample. Two rigidly mounted corotating glass disks with suitable arrays of holes ensure that adjacent foci illuminate the sample at different time points. Recordings of biological specimens demonstrate elimination of out-of-focus haze for densely packed foci and concomitant substantial improvement of contrast and resolution.

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

Time multiplexing and parallelization in multifocal multiphoton microscopy

We investigate the imaging properties of high-aperture multifocal multiphoton microscopy on the basis of diffraction theory. Particular emphasis is placed on the relationship between the sectioning property and the distance between individual foci. Our results establish a relationship between the degree of parallelization and the axial resolution for both two- and three-photon excitation. In addition, we show quantitatively that if a matrix of temporal delays is inserted between the individual foci, it is, for the first time to our knowledge, possible to solve the classical conflict between the light budget and the sectioning property in three-dimensional microscopy and to provide a virtually unlimited density of foci at best axial resolution.