Density and function of actin-microdomains in healthy and NF1 deficient osteoclasts revealed by the combined use of atomic force and stimulated emission depletion microscopy

Actin and myosins (IIA, IIB, and X) generate mechanical forces in osteoclasts that drive functions such as migration and membrane trafficking. In neurofibromatosis, these processes are perturbed due to a mutation in neurofibromatosis type 1 (NF1) gene. This mutation leads to generation of hyperactive bone-resorbing osteoclasts that increases incidence of skeletal dysplasia e.g. early-onset osteoporosis in patients suffering from neurofibromatosis. To study the density and function of actin clusters in mutated cells we introduce a new approach for combined use of a stimulated emission depletion (STED) microscope with an atomic force microscope (AFM). We resolved actin-cores within actin-microdomains at four typical structures (podosome-belt, podosome raft, actin patches, and sealing zone) for osteoclasts cultured on bone as well as on glass. Densities of actin-cores in these structures were higher on bone than on glass, and the nearest neighbor distances were shortest in sealing zones, where also an accumulation of vesicular material was observed at their center. In NF1 deficient osteoclasts, the clustering was tighter and there was also more vesicular material accumulated inside the sealing zone. Using the STED-AFM system, we measured the condensation of the actin structures in real-time after a bone-coated cantilever was placed in contact with a differentiated osteoclast and found that the condensation of actin was initiated at 40 min, after sufficient local actin concentration was reached. A functional implication of the less dense clustering in NF1 deficient cells was that the adhesion of these cells was less specific for bone. The data and new methodologies presented here build a foundation for establishing novel actomyosin dependent mechanisms during osteoclast migration and resorption.</p

Density and function of actin-microdomains in healthy and NF1 deficient osteoclasts revealed by the combined use of atomic force and stimulated emission depletion microscopy

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Kilohertz retinal FF-SS-OCT and flood imaging with hardware-based adaptive optics.

A retinal imaging system was designed for full-field (FF) swept-source (SS) optical coherence tomography (OCT) with cellular resolution. The system incorporates a real-time adaptive optics (AO) subsystem and a very high-speed CMOS sensor, and is capable of acquiring volumetric images of the retina at rates up to 1 kHz. While digital aberration correction (DAC) is an attractive potential alternative to AO, it has not yet been shown to provide resolution allowing visualization of cones in the fovea, where early detection of functional deficits is most critical. Here we demonstrate that FF-SS-OCT with hardware AO permits resolution of foveal cones, imaged at eccentricities of 1° and 2°, with volume rates adequate to measure light-evoked changes in photoreceptors. With the reference arm blocked, the system can operate as a kilohertz AO flood illumination fundus camera with adjustable temporal coherence and is expected to allow measurement of light-evoked changes caused by common path interference in photoreceptor outer segments (OS). In this paper, we describe the system's optical design, characterize its performance, and demonstrate its ability to produce images of the human photoreceptor mosaic

Non-invasive in vivo angiography of the human eye with Doppler Optical Coherence Tomography

Introduction Optical coherence tomography (OCT) uses laser interferometry for non-invasive cross-sectional imaging of tissues with micrometer resolution. This technology is therefore ideal to visualize the micro-structures of the human retina and choroid in vivo. Additionally blood flow can be detected from Doppler frequency shifts in the OCT signal over time, which are caused by moving particles in flowing blood. In this study we investigated if these Doppler shifts can be used to create angiograms of the retina and choroid. Methods An experimental OCT system was constructed based on a 1040 nm swept laser source. A healthy volunteer was imaged over a retinal area of 6.0 × 7.9 mm 2 (20º × 26º). Doppler shifts were evaluated by measuring each location twice and were calculated from phase changes within the OCT signals. Angiograms of the vasculature were created by integration of the phase changes over depth. Results The retinal angiogram (Fig. 1(A)) shows blood vessels (in white) down to the capillary level and visualizes clearly the avascular zone of the fovea and the entrance and exit of vessels through the optic disc. The choroidal angiogram (Fig. 1(B)) shows a dense network of large vessels below the retina. Conclusions Doppler OCT can produce high-resolution angiograms of the retina and choroid

Dual wavelength Scanning Light Ophthalmoscope with concentric circle scanning

Abstract Purpose :Multispectral imaging helps in gathering important physiological parameters about the retina. We present a novel and compact scanning light ophthalmoscope (SLO) using a digital micromirror device (DMD) capable of imaging the retina at 7 Hz at two different wavelengths with a maximum 20° × 20° field of view (FOV). Methods :The dual-wavelength SLO (Fig.1) used DMD to create concentric circle scanning on the retina. The concentric circles were centred around the fovea and provided fixation. By shifting the centre of the circles to different locations on the DMD, we imaged different regions of the retina. An annulus was placed in conjugate to the pupil plane in the illumination arm to create an annular illumination on the cornea. In the detection arm, a circular aperture was used to block corneal reflections and pass only the signal reflected from the retina onto the camera. Blocking the corneal reflections reduced the background and increased the signal to noise ratio. Polarisation optics were used to discard the stray reflections within the system. Virtual pinholes were implemented in the digital to create confocal images (Heintzmann et al, 2006). To demonstrate the capabilities of our system, we imaged the right eye of a healthy volunteer with a DMD pattern projection speed of 140 Hz and a fill-factor of 1/20. We used 660 nm and 810 nm illuminations to record confocal images. Results :Fig. 2A shows the fundus photograph of the subject. Fig. 2B & 2C shows the images of the macular region with the fovea in the centre imaged using 810 nm and 660 nm. Since we used polarisation optics, the macular bowtie structure is visible in Figs. 2B & 2C. The optic nerve head region is shown in Figs. 2D & 2E for both wavelengths. Blood vessels in the perifoveal inferior region were also imaged (Fig.2F & 2G). These images show high contrast details of the retina with big and small blood vessels at two different wavelengths. Conclusions :We have demonstrated multispectral retinal imaging using a DMD to create high contrast retinal images. The DMD enables fixating the eye to different locations allowing us to image different parts of peri- and parafoveal regions. This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017. Figure1: Optical layout of the system Figure 2: Imaging in the right eye of a healthy volunteer (A) : Fundus photo. (B-G) : Confocal images of different peri- and para-foveal regions imaged by 810 nm and 660 nm denoted by coloured dotted circles. Scale bar is 5 degrees

Parallel line scanning ophthalmoscope for retinal imaging

Purpose: To visualize retinal structures using a newly developed parallel line scanning ophthalmoscope (PLSO). Methods: A PLSO was built using a digital micromirror device (DMD) instead of traditional scanning mirrors to scan lines over the field of view (FOV). The DMD consists of 912 × 1140 micromirrors which can be individually switched on/off based on a programmed binary pattern. By switching on multiple (parallel) two-element wide lines in the DMD, the corresponding lines on the retina are imaged on a CMOS camera. After acquisition of each frame, the micromirrors are turned off and the mirrors for the next set of adjacent lines are turned on. This is repeated until the whole FOV is imaged. Confocal images are generated from the data by subtracting the maximum and minimum intensity values for each pixel in the sequence. The fovea and optic nerve head (ONH) of a healthy subject were imaged using 10º × 10º FOV at 100 Hz with 7 parallel lines resulting in a full image frame rate of 1.4 fps. The images were acquired through a dark-adapted pupil without any dilatation. The acquired data were processed, as mentioned earlier, into confocal images; but also non-confocal images were obtained by averaging all frames. Results: Figure 1A shows the imaged areas. In the non-confocal images (Fig. 1B&C), the corneal scattering is dominant and makes the retinal structures covered in haze. In the confocal images (Fig. 1D&E), confocality and contrast are improved. The foveal avascular zone and smaller blood vessels are visible in the fovea image (Fig. 1D). Also the quality of the ONH image is improved and many of the main features can be distinguished such as small blood vessels (Fig. 1E). Conclusions: The PLSO provided high contrast images of the fovea and ONH and detailed retinal structures could be observed. The DMD eliminates moving parts from the system and exposure time for each frame is potentially shorter than in full-field imaging, which reduces intra-frame motion. In retinal imaging, such a setup will provide better images because higher imaging speeds reduce motion artifacts

In vivo retinal imaging for fixational eye motion detection using a high-speed digital micromirror device (DMD)-based ophthalmoscope

Retinal motion detection with an accuracy of 0.77 arcmin corresponding to 3.7 µm on the retina is demonstrated with a novel digital micromirror device based ophthalmoscope. By generating a confocal image as a reference, eye motion could be measured from consecutively measured subsampled frames. The subsampled frames provide 7.7 millisecond snapshots of the retina without motion artifacts between the image points of the subsampled frame, distributed over the full field of view. An ophthalmoscope pattern projection speed of 130 Hz enabled a motion detection bandwidth of 65 Hz. A model eye with a scanning mirror was built to test the performance of the motion detection algorithm. Furthermore, an in vivo motion trace was obtained from a healthy volunteer. The obtained eye motion trace clearly shows the three main types of fixational eye movements. Lastly, the obtained eye motion trace was used to correct for the eye motion in consecutively obtained subsampled frames to produce an averaged confocal image correct for motion artefacts

Optoretinogram: optical measurement of human cone and rod photoreceptor responses to light.

Noninvasive, objective measurement of rod function is as significant as that of cone function, and for retinal diseases such as retinitis pigmentosa and age-related macular degeneration, rod function may be a more sensitive biomarker of disease progression and efficacy of treatment than cone function. Functional imaging of single human rod photoreceptors, however, has proven difficult because their small size and rapid functional response pose challenges for the resolution and speed of the imaging system. Here, we describe light-evoked, functional responses of human rods and cones, measured noninvasively using a synchronized adaptive optics optical coherence tomography (OCT) and scanning light ophthalmoscopy (SLO) system. The higher lateral resolution of the SLO images made it possible to confirm the identity of rods in the corresponding OCT volumes

Imaging of optic nerve head pore structure with motion corrected deeply penetrating OCT using tracking SLO

Purpose To remove the eye motion and stabilize the optical frequency domain imaging (OFDI) system for obtaining high quality images of the optic nerve head (ONH) and the pore structure of the lamina cribrosa. Methods An optical coherence tomography (OCT) instrument was combined with an active eye tracking system to compensate for eye motion in OCT imaging. The OCT system was a phase-stabilized deeply penetrating OFDI system operating at center wavelength of 1040 nm and the eye tracker was an 840 nm scanning laser ophthalmoscope (SLO). Retinal tracking was performed using real-time analysis of the distortions within SLO frames. OFDI had axial resolution of 4.8 µm (6.5 µm in air) and the theoretical spot-size on the retina was 13.7 µm. Eye motion was reported at a rate of 960 Hz and motion signals were inverted to correction signals and used to keep the OCT scanning grid locked on the same retinal area throughout the measurement. In the case of a tracking lock failure (e.g. blink or large saccade), the tracker signaled the OFDI system to rescan corrupted B-scans immediately stepping back 10 B-scans and holding the position until signal was valid again. The achieved tracking bandwidth was 32 Hz due to an internal time lag of the hardware. The ONH of a healthy volunteer was imaged over an area of 2.7 × 2.7 mm (8.8°) using 700 A-scans/B-scan. To visualize the benefit of the tracking, each acquired B-scan in a volume dataset (total of 700 B-scans) was integrated over depth to create an enface image of the ONH. Results The ONH was successfully imaged with negligible artifacts from eye motion (Fig. 1). On the left side, the whole dataset is seen including the duplicate corrupted B-scans. The corrupted B-scans were then removed in post-processing, thus leaving the undistorted duplicates untouched. The measured residual motion in the OCT B-scans was 0.32 arcmin (~1.6 µm) in a human eye. Four volumes from the same location were registered together to visualize the lamina cribrosa throughout the different depth slices of the eye (Fig. 2). The pore structure was clearly visible up to 430 um from the bottom of the ONH cup. Conclusions It is possible to obtain high quality OCT images from ONH and lamina cribrosa by compensating the eye motion during the measurements

Media 2: Angiography of the retina and the choroid with phase-resolved OCT using interval-optimized backstitched B-scans

Originally published in Optics Express on 27 August 2012 (oe-20-18-20516