Orthogonal-plane Fluorescence Optical Sectioning Research Articles

A qualitative comparison of 3D visualization in Xenopus laevis using a traditional method and a non-destructive method

Many tools are currently available to investigate and visualize soft and hard tissues in animals both in high-resolution and three dimensions. The most popular and traditional method is based on destructive histological techniques. However, these techniques have some specific limitations. In order to avoid those limitations, various non-destructive approaches have surfaced in the last decades. One of those is micro-CT-scanning. In the best conditions, resolution achieved in micro-CT currently approaches that of standard histological protocols. In addition to bone, soft tissues can also be made visible through micro-CT-scanning. However, discriminating between structures of the same tissue and among different tissue types remains a challenge. An alternative approach, which has not yet been explored to its full potential for comparative anatomy studies, is Orthogonal-Plane Fluorescence Optical Sectioning (OPFOS) microscopy or tomography, also known as (Laser) Light Sheet based Fluorescence Microscopy (LSFM). In this study, we compare OPFOS with light microscopy, applying those techniques to the model organism Xenopus laevis. The potential of both methods for discrimination between different types of tissues, as well as different structures of the same tissue type, is tested and illustrated. Since the histological sections provided a better resolution, adjacent structures of the same tissue type could be discerned more easily compared to our OPFOS images. However, we obtained a more naturally-shaped 3D model of the musculoskeletal system of Xenopus laevis with OPFOS. An overview of the advantages and disadvantages of both techniques is given and their applicability for a wider scope of biological research is discussed.

Three-Dimensional Imaging and Modeling of Anatomic Structures, Sectional and Radiological Anatomy, and Staining Techniques

The first three-dimensional (3D) reconstruction was performed by Born (1883) and His (1885) using serial sections. Since then, a variety of reconstruction techniques have been developed, and the construction of physical models has become important in anatomy. The first computer-aided 3D reconstruction was achieved by Glaser and Van der Loos in 1965. With the improvements in both computer hardware and software tools, computerized modelling of anatomical structures has become very useful for visualizing complex 3D forms. Three-dimensional visualization of various microanatomic structures using special preparation and staining methods is important. Devices used for this purpose such as MicroCT, microMRI, SEM, and Confocal microscopy and so forth. are development parallel to the technological advances and give excellent 3D images, and it allows a better understanding of gross and microanatomic structures. On the other hand, first studies on cross-sectional anatomy date back to the 16th century. The widespread use of cross-sectional imaging techniques such as CT and MRI has gained an increasing importance in sectional anatomy. The most known cadaveric cross-sectional study, the visible human project, is an outgrowth of the National Library Medicine. In this special issue, we reviewed and edited seven articles on three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. K. K. Schmid (University of Nebraska, USA) Marx and Samal (University of Nebraska-Lincoln, USA) discussed three-dimensional regression, a technique that can be used for mapping images and shapes that are represented by sets of three-dimensional landmark coordinates, for comparing and mapping 3D anatomical structures in their research paper. Y. Hoshino and coworkers (University of Pittsburgh, USA) reviewed previous research about image analysis of the anterior cruciate ligament (ACL) anatomy and its application to ACL reconstruction surgery. They concluded that three-dimensional image analysis of the ACL anatomy and its application to the navigation system is becoming more prevalent and reliable for advancing the anatomic studies related to the native ACL and the ACL reconstruction procedure. A. Arencibia et al. (Veterinary Faculty, University of Las Palmas de Gran Canaria) investigated imaging features of the temporomandibular joint (TMJ) using computed tomography and magnetic resonance imaging in two normal camels. They stated in conclusion that CT is an excellent method for the detailed assessment of the bony structures, and MRI is a valid imaging modality for the evaluation of the soft tissues. Processing of CT and MR images allowes to appreciate different bone structures and soft tissues of the TMJ, assisting in the interpretation of the images. J. A. N. Buytaert and colleagues (University of Antwerp and Ghent University, Belgium) discussed the first implementation of (laser) light-sheet-based fluorescence microscopy (LSFM) to image biomedical tissue in three-dimensional orthogonal plane fluorescence optical sectioning microscopy (OPFOS). They have shown with several applications that the OPFOS (and derived) methods are a valuable addition for sectional imaging and three-dimensional modeling of anatomic structures. The review paper entitled “Ultrahigh voltage electron microscopy (UHVEM) links neuroanatomy and neuroscience/neuroendocrinology” by H. Sakamoto (Okayama University, Japan) and M. Kawata (Kyoto Prefectural University of Medicine, Japan) stressed that UHVEM and light microscopy are useful and powerful tools for studying molecular and/or chemical neuroanatomy at the ultrastructural level. D. R. Marker et al. (The Johns Hopkins Hospital and University School of Medicine, Weill Cornell Medical College, USA) discussed strategic improvements for gross anatomy web-based teaching in their research paper. To better meet the expectations of gross anatomy students, electronic radiology teaching files for first-year coursework were organized into a web site. The web site was custom designed to provide material that directly correlated to the gross anatomy dissection and lectures. Quick links provided sets of images grouped by anatomic location. Their findings suggest that a well-organized web portal can provide a user-friendly, valuable educational resource for medical students who are studying gross anatomy. Finally, Herber and coworkers (University of California, USA) in their study entitled “Imaging an adapted dentoalveolar complex” used micro-X-ray computed tomography combined with 3D modeling using image processing, scanning electron microscopy, fluorochrome labeling, conventional histology (H&E, TRAP), and immunohistochemistry (RANKL, OPN) to elucidate the dynamic nature of bone, the periodontal ligamentspace, and cementum in the rat periodontium. They concluded that studies focusing on a functional rat dentoalveolar complex should acknowledge the baseline phenomena of bone-PDL-cementum adaptation, especially in regards to the physiological distal drift, before additional experimental variables are imposed. We hope that this special issue will contribute as a stimulus for further research into three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. We are very grateful to the contributing authors for their scientific contributions to this special issue. We also thank the reviewers who spend their valuable time and thoughts on each paper. We appreciate the Hindawi Publishing Corporation for their help in the guidance of the procedure. Tuncay Peker Nadir Gulekon Ilkan Tatar Levent Sarikcioglu David Kachlik

The OPFOS Microscopy Family: High-Resolution Optical Sectioning of Biomedical Specimens

We report on the recently emerging (laser) light-sheet-based fluorescence microscopy field (LSFM). The techniques used in this field allow to study and visualize biomedical objects nondestructively in high resolution through virtual optical sectioning with sheets of laser light. Fluorescence originating in the cross-section of the sheet and sample is recorded orthogonally with a camera. In this paper, the first implementation of LSFM to image biomedical tissue in three dimensions—orthogonal-plane fluorescence optical sectioning microscopy (OPFOS)—is discussed. Since then many similar and derived methods have surfaced, (SPIM, ultramicroscopy, HR-OPFOS, mSPIM, DSLM, TSLIM, etc.) which we all briefly discuss. All these optical sectioning methods create images showing histological detail. We illustrate the applicability of LSFM on several specimen types with application in biomedical and life sciences.

Meeting report: First light sheet based fluorescence microscopy workshop

In Jena, at the turn of the century, Richard Zsig-mondy (1865–1929), working together with thephysicist Heinrich Siedentopf, had the brilliantidea to use a light sheet to illuminate from the sidethe colloid solution they were investigating [1].From there the idea of light sheet illumination van-ished from the scientific spotlight until it was re-vived it in the 90’s as Orthogonal-Plane Fluores-cence Optical Sectioning (ORFOS) [2] and thetaconfocal microscopy [3]. At the beginning of thenew millennium, Ernst Stelzer and colleagues de-veloped Selective Plane Illumination Microscopy(SPIM) and applied it to live imaging of model or-ganism embryos [4,5].Since then the light-sheet il-lumination approach proved instrumental for awide variety of biological disciplines ranging frommarine biology [6], to fundamental developmental[7,8] and cell biology research questions [9,10].Yetthe technology remains largely confined to the lab-oratories that orchestrated its revival and the ben-efits of light sheet illumination are inaccessible tothe broad scientific community. It was the purposeof the First Light Sheet based Fluorescence Mi-croscopy workshop to bring together the key play-ers in the light sheet technology and to establish ef-fective communication among them that will hope-fully lead to better integration of light sheet mi-croscopy into scientific mainstream. It is symbolicthat the workshop took place at the Max Planck In-stitute of Molecular Cell Biology and Genetics(MPI-CBG) in Dresden only some 100 miles awayfrom Jena where it all started.The workshop aimed at founding a communityand to encourage communication and sharing be-tween the different groups developing, using orsimply interested in this technology for their re-search. In preparation for the workshop we estab-lished a Wiki where every invited participant coulddescribe his research and sign up for a presenta-tion in one of the sessions (https://spim.mpi-cbg.de).This self-organizing approach worked verywell, as most of the participants were in fact pre-senting in the session of their choice and all speak-ers received equal amount of time to present theirdata.The presentations were divided in three mainsections:

Three-dimensional reconstruction of the pigeon inner ear

Three-dimensional reconstructions of the inner ear of the pigeon (Columba livia domestica), from two-dimensional images, obtained with (conventional) light microscopy or orthogonal-plane fluorescence optical sectioning (OPFOS), are presented. The results are compared with available information on the dimensions and orientation of the semicircular canals.

Tomographic imaging of macroscopic biomedical objects in high resolution and three dimensions using orthogonal-plane fluorescence optical sectioning

A new optical-fluorescence microscopy technique, called HR-OPFOS, is discussed and situated among similar OPFOS-implementations. OPFOS stands for orthogonal-plane fluorescence optical sectioning and thus is categorized as a laser light sheet based fluorescence microscopy method. HR-OPFOS is used to make tomographic recordings of macroscopic biomedical specimens in high resolution. It delivers cross sections through the object under study with semi-histological detail, which can be used to create three-dimensional computer models for finite-element modeling or anatomical studies. The general innovation of this class of microscopy setup consists of the separation of the illumination and observation axes, but now in our setup combined with focal line scanning to improve sectioning resolution. HR-OPFOS is demonstrated on gerbil hearing organs and on mouse and bird brains. The necessary specimen preparation is discussed.

Three‐dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 microm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.

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.

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.

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.