Fluorescence Optical Sectioning Research Articles

Morphology and function of Bast’s valve: additional insight in its functioning using 3D-reconstruction

The utriculo-endolymphatic valve was discovered by Bast in 1928. The function of Bast’s valve is still unclear. By means of orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy 3D-reconstructions of the valve and its surrounding region are depicted. The shape of the duct at the utricular side is that of a flattened funnel. In the direction of the endolymphatic duct and sac this funnel runs into a very narrow duct. The valve itself has a rigid ‘arch-like’ configuration. The opposing thin, one cell-layer thick, utricular membrane is highly compliant. We propose that opening and closure of the valve occurs through movement of the flexible base/utricular membrane away from and toward the relatively rigid valve lip.

In Vivo Estimates of the Position of Advanced Bionics Electrode Arrays in the Human Cochlea

A new technique for determining the position of each electrode in the cochlea is described and applied to spiral computed tomography data from 15 patients implanted with Advanced Bionics HiFocus I, Ij, or Helix arrays. ANALYZE imaging software was used to register 3-dimensional image volumes from patients' preoperative and postoperative scans and from a single body donor whose unimplanted ears were scanned clinically, with micro computed tomography and with orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy. By use of this registration, we compared the atlas of OPFOS images of soft tissue within the body donor's cochlea with the bone and fluid/ tissue boundary available in patient scan data to choose the midmodiolar axis position and judge the electrode position in the scala tympani or scala vestibuli, including the distance to the medial and lateral scalar walls. The angular rotation 0 degrees start point is a line joining the midmodiolar axis and the middle of the cochlear canal entry from the vestibule. The group mean array insertion depth was 477 degrees (range, 286 degrees to 655 degrees). The word scores were negatively correlated (r = -0.59; p = .028) with the number of electrodes in the scala vestibuli. Although the individual variability in all measures was large, repeated patterns of suboptimal electrode placement were observed across subjects, underscoring the applicability of this technique.

In Vivo Estimates of the Position of Advanced Bionics Electrode Arrays in the Human Cochlea

Objectives:A new technique for determining the position of each electrode in the cochlea is described and applied to spiral computed tomography data from 15 patients implanted with Advanced Bionics HiFocus I, Ij, or Helix arrays.Methods:ANALYZE imaging software was used to register 3-dimensional image volumes from patients' preoperative and postoperative scans and from a single body donor whose unimplanted ears were scanned clinically, with micro computed tomography and with orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy. By use of this registration, we compared the atlas of OPFOS images of soft tissue within the body donor's cochlea with the bone and fluid/tissue boundary available in patient scan data to choose the midmodiolar axis position and judge the electrode position in the scala tympani or scala vestibuli, including the distance to the medial and lateral scalar walls. The angular rotation 0° start point is a line joining the midmodiolar axis and the middle of the cochlear canal entry from the vestibule.Results:The group mean array insertion depth was 477° (range, 286° to 655°). The word scores were negatively correlated (r = −0.59; p = .028) with the number of electrodes in the scala vestibuli.Conclusions:Although the individual variability in all measures was large, repeated patterns of suboptimal electrode placement were observed across subjects, underscoring the applicability of this technique.

Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution

Several well-established techniques are available to obtain 3-D image information of biomedical specimens, each with their specific advantages and limitations. Orthogonal plane fluorescence optical sectioning (OPFOS), or selective plane illumination microscopy (SPIM), are additional techniques which, after adequate specimen preparation, produce high quality, autoaligned sectional images in nearly real time, of bone as well as soft tissue. Up until now, slicing resolutions down to 14 microm have been obtained. We present a high resolution (HR) OPFOS method, which delivers images that approach the quality of histological sections. With our HROPFOS technique, we achieve in-plane resolutions of 1 microm and a slicing resolution of 2 microm. A region of interest within an intact and much larger object can be imaged without problems, and as the optical technique is nondestructive, the object can be measured in any slicing direction. We present quantitative measurements of resolution. A 3-D model reconstructed from our HROPFOS data is compared to SEM results, and the technique is demonstrated with section images and 3-D reconstructions of middle ear specimens.

The relationship of the round window membrane to the cochlear aqueduct shown in three-dimensional imaging

The round window membrane and cochlear aqueduct complex in the guinea pig are reconstructed with 3D-imaging, using orthogonal plane fluorescence optical sectioning (OPFOS). The 3D-images show that the periotic duct and the aqueduct are connected to a pouch-like extension of the round window. The function of this may be regulation of aqueduct flow resistance under the influence of a pressure difference between inner ear fluid and middle ear.

Wave front engineering for microscopy of living cells

A new method to perform simultaneously three dimensional optical sectioning and optical manipulation is presented. The system combines a multi trap optical tweezers with a video microscope to enable axial scanning of living cells while maintaining the trapping configuration at a fixed position. This is achieved compensating the axial movement of the objective by shaping the wave front of the trapping beam with properly diffractive optical elements displayed on a computer controlled spatial light modulator. Our method has been validated in three different experimental configurations. In the first, we decouple the position of a trapping plane from the axial movements of the objective and perform optical sectioning of a circle of beads kept on a fixed plane. In a second experiment, we extend the method to living cell microscopy by showing that mechanical constraints can be applied on the dorsal surface of a cell whilst performing its fluorescence optical sectioning. In the third experiment, we trapped beads in a three dimensional geometry and perform, always through the same objective, an axial scan of the volume delimited by the beads.

Axial gain resolution in optical sectioning fluorescence microscopy by shaded-ring filters

We present a new family of pupil masks to control the axial component of the intensity distribution in the focal region of tightly focused light fields. The filters, which consist of a circular clear pupil with a single shaded ring, allow to control the width of the central lobe of the axial spot together with the residual sidelobes energy. The filters can be applied to improve the optical sectioning capacity of different scanning microscopes.

Three-ring filters increase the effective NA up to 1.46 in optical sectioning fluorescence microscopy

Single-photon fluorescence confocal microscopy techniques can be combined with the use of specific binary filters in order to increase their optical sectioning capability. We present a novel class of axially super-resolving binary pupil filters specially designed to reach this aim. These filters let us to obtain a relevant compression of the z-response together with the reduction of the photo-bleaching effect typically inherent to apodization techniques. The fact of joining both the three-ring filters we propose in the illumination path, and the confocal detection gives rise to an important effective increase of lenses of effective numerical aperture.

Three-dimensional modelling of the middle-ear ossicular chain using a commercial high-resolution X-ray CT scanner.

The quantitative measurement of the three-dimensional (3-D) anatomy of the ear is of great importance in the making of teaching models and the design of mathematical models of parts of the ear, and also for the interpretation and presentation of experimental results. This article describes how we used virtual sections from a commercial high-resolution X-ray computed tomography (CT) scanner to make realistic 3-D anatomical models for various applications in our middle-ear research. The important problem of registration of the 3-D model within the experimental reference frame is discussed. The commercial X-ray CT apparatus is also compared with X-ray CT using synchrotron radiation, with magnetic resonance microscopy, with fluorescence optical sectioning, and with physical (histological) serial sections.

Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy

Orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy was developed for the purpose of making quantitative measurements of the intact mammalian cochlea and to facilitate 3D reconstructions of complex features. A new version of this imaging apparatus was built with a specimen chamber designed to accommodate samples as large as the intact guinea pig bulla. This method left the cochlear connections with the vestibular system and with the ossicles of the middle ear undisturbed, providing views within the cochlea with no breaches of its structural integrity. Since the features within the bulla were not physically touched during the preparation process, the risk of damage was minimized, and were imaged in relatively pristine condition with spatial resolution to 16 μm. A description of the imaging method and specimen preparation procedure is presented, as are images of features from the cochlea, ossicles, and vestibular system.

F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe.

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase, the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ss-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result, Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

Spatiotemporal Analysis of Flow-Induced Intermediate Filament Displacement in Living Endothelial Cells

The distribution of hemodynamic shear stress throughout the arterial tree is transduced by the endothelium into local cellular responses that regulate vasoactivity, vessel wall remodeling, and atherogenesis. Although the exact mechanisms of mechanotransduction remain unknown, the endothelial cytoskeleton has been implicated in transmitting extracellular force to cytoplasmic sites of signal generation via connections to the lumenal, intercellular, and basal surfaces. Direct observation of intermediate filament (IF) displacement in cells expressing green fluorescent protein-vimentin has suggested that cytoskeletal mechanics are rapidly altered by the onset of fluid shear stress. Here, restored images from time-lapse optical sectioning fluorescence microscopy were analyzed as a four-dimensional intensity distribution function that represented IF positions. A displacement index, related to the product moment correlation coefficient as a function of time and subcellular spatial location, demonstrated patterns of IF displacement within endothelial cells in a confluent monolayer. Flow onset induced a significant increase in IF displacement above the nucleus compared with that measured near the coverslip surface, and displacement downstream from the nucleus was larger than in upstream areas. Furthermore, coordinated displacement of IF near the edges of adjacent cells suggested the existence of mechanical continuity between cells. Thus, quantitative analysis of the spatiotemporal patterns of flow-induced IF displacement suggests redistribution of intracellular force in response to alterations in hemodynamic shear stress acting at the lumenal surface.

Studies on the structure of sperm heads of Eledone cirrhosa by means of CLSM linked to bioimage-oriented devices.

We have used a confocal laser scanning optical microscope imaging device and a bioimage-oriented workstation equipped for augmented reality to study the helical sperm head of the octopus Eledone cirrhosa. This approach allows us to study different complex organisational motifs due to the spatial arrangement of linear helical structures. We consider this helical specimen an enlarged copy of one of the most important biostructures governing cell functioning such as chromatin-DNA. Moreover, this very same sample is made of highly compacted chromatin that can be studied at higher resolution, i.e., by means of scanning force microscopy. Fluorescence optical sectioning has been used to enter the spatial organisation. Three-dimensional images of single, twisted, and folded fibers are shown.

All-solid-state ultrafast lasers facilitate multiphoton excitation fluorescence imaging

Improvements in ultrafast laser technology have enabled a new excitation mode for optical sectioning fluorescence microscopy: multiphoton excitation fluorescence imaging. The primary advantages of this technique over laser scanning confocal imaging derive from the localized excitation volume; additional advantages accrue from the longer wavelength of the excitation source. Recent advances in all-solid-state, ultrafast (subpicosecond) laser technology should allow the technique to gain widespread use as a commercial instrument. In this paper, we review: optical sectioning fluorescence microscopy, multiphoton excitation fluorescence laser scanning microscopy, developments in laser physics which have enabled all-solid-state lasers to be used as excitation sources for multiphoton excitation fluorescence imaging, and provide current data for all-solid-state ultrafast lasers. A direct comparison between confocal (488 nm) imaging and two-photon excitation (1047 nm) imaging of a mouse brain slice stained with the lipophilic dye FM4-64 shows two-photon imaging can provide usable images more than twice as deep as confocal imaging. Multi-mode images (both two- and three-photon excitation) are presented for fixed and living cells as examples of multiphoton excitation fluorescence imaging applied to developmental biology. Also, a comparison of the axial resolution of our system is presented for confocal imaging (488 nm) and two-photon imaging (1047 nm) with and without a confocal pinhole aperture.

Three-dimensional reconstruction of the cochlea from two-dimensional images of optical sections

This paper describes a methodology for three-dimensional (3D) computer-aided reconstruction of the guinea pig cochlea using orthogonal-plane fluorescence optical sectioning. Specimens are sectioned optically, allowing them to remain intact during observation. Equations to correct the data for specimen translation and rotation are developed and 3D reconstructions of the scala tympani, round window membrane, and cochlear aqueduct are presented. The error associated with the reconstruction is estimated to be <19 μm.

Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.

An imaging technique called orthogonal-plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three-dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high-resolution images of optical sections of an intact, excised guinea-pig cochlea. The lateral and axial resolutions of the images were 10 and 26 microns, respectively, within a 1.5-mm field of view.