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.

Moiré profilometry using liquid crystals for projection and demodulation.

A projection moiré profilometer is presented in which both projection and optical demodulation are realized with liquid crystal light modulators. The computer generated grids, realized on thin film transistor matrices, allow phase-stepping and discrete grid averaging without the need for any mechanically moving component. Spatial line pitch and phase steps can thus be readily adjusted to suit the measurement precision and object geometry. The device is able to perform topographic measurements with a height resolution of 15 microm on every pixel of the recording device.

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.

Quasi-static transfer function of the rabbit middle ear' measured with a heterodyne interferometer with high-resolution position decoder.

Due to changes in ambient pressure and to the gas-exchange processes in the middle ear (ME) cavity, the ear is subject to ultra-low-frequency pressure variations, which are many orders of magnitude larger than the loudest acoustic pressures. Little quantitative data exist on how ME mechanics deals with these large quasi-static pressure changes and because of this lack of data, only few efforts could be made to incorporate quasi-static behavior into computer models. When designing and modeling ossicle prostheses and implantable ME hearing aids, the effects of large ossicle movements caused by quasi-static pressures should be taken into account. We investigated the response of the ME to slowly varying pressures by measuring the displacement of the umbo and the stapes in rabbit with a heterodyne interferometer with position decoder. Displacement versus pressure curves were obtained at linear pressure change rates between 200 Pa/s and 1.5 kPa/s, with amplitude +/-2.5 kPa. The change in stapes position associated with a pressure change is independent of pressure change rate (34 microm peak-to-peak at +/-2.5 kPa). The stapes displacement versus pressure curves are highly nonlinear and level off for pressures beyond +/-1 kPa. Stapes motion shows no measurable hysteresis at 1.5 kPa/s, which demonstrates that the annular ligament has little viscoelasticity. Hysteresis increases strongly at the lowest pressure change rates. The stapes moves in phase with the umbo and with pressure, but the sense of rotation of the hysteresis loop of stapes is phase inversed. Stapes motion is not a simple lever ratio mimic of umbo motion, but is the consequence of complex changes in ossicle joints and ossicle position. The change in umbo position produced by a +/-2.5 kPa pressure change decreases with increasing rate from 165 microm at 200 Pa/s to 118 microm at 1.5 kPa/s. Umbo motion already shows significant hysteresis at 1.5 kPa/s, but hysteresis increases further as pressure change rate decreases. We conclude that in the quasi-static regime, ossicle movement is not only governed by viscoelasticity, but that other effects become dominant as pressure change rate decreases below 1 kPa/s. The increasing hysteresis can be caused by increasing friction as speed of movement decreases, and incorporating speed-dependent friction coefficients will be essential to generate realistic models of ossicle movements at slow pressure change rates.

Details of human middle ear morphology based on micro-CT imaging of phosphotungstic acid stained samples.

A multitude of morphological aspects of the human middle ear (ME) were studied qualitatively and/or quantitatively through the postprocessing and interpretation of micro-CT (micro X-ray computed tomography) data of six human temporal bones. The samples were scanned after phosphotungstic acid staining to enhance soft-tissue contrast. The influence of this staining on ME ossicle configuration was shown to be insignificant. Through postprocessing, the image data were converted into surface models, after which the approaches diverged depending on the topics of interest. The studied topics were: the ME ligaments; morphometric and mechanical parameters of the ossicles relating to inertia and the ossicular lever arm ratio; the morphology of the distal incus; the contact surface areas of the tympanic membrane (TM) and of the stapes footplate; and the thickness of the TM, round window of the cochlea, ossicle joint spaces, and stapedial annular ligament. Some of the resulting insights are relevant in ongoing discussions concerning ME morphology and mechanical functions, while other results provide quantitative data to add to existing data. All findings are discussed in the light of other published data and many are relevant for the construction of mechanical finite element simulations of the ME.

MicroCT versus sTSLIM 3D imaging of the mouse cochlea.

We made a qualitative and quantitative comparison between a state-of-the-art implementation of micro-Computed Tomography (microCT) and the scanning Thin-Sheet Laser Imaging Microscopy (sTSLIM) method, applied to mouse cochleae. Both imaging methods are non-destructive and perform optical sectioning, respectively, with X-rays and laser light. MicroCT can be used on fresh or fixed tissue samples and is primarily designed to image bone rather than soft tissues. It requires complex back-projection algorithms to produce a two-dimensional image, and it is an expensive instrument. sTSLIM requires that a specimen be chemically fixed, decalcified, and cleared; but it produces high-resolution images of soft and bony tissues with minimum image postprocessing and is less expensive than microCT. In this article, we discuss the merits and disadvantages of each method individually and when combined.

Full-field thickness distribution of human tympanic membrane obtained with optical coherence tomography.

The full-field thickness distribution, three-dimensional surface model and general morphological data of six human tympanic membranes are presented. Cross-sectional images were taken perpendicular through the membranes using a high-resolution optical coherence tomography setup. Five normal membranes and one membrane containing a pathological site are included in this study. The thickness varies strongly across each membrane, and a great deal of inter-specimen variability can be seen in the measurement results, though all membranes show similar features in their respective relative thickness distributions. Mean thickness values across the pars tensa ranged between 79 and 97 µm; all membranes were thinnest in the central region between umbo and annular ring (50-70 µm), and thickness increased steeply over a small distance to approximately 100-120 µm when moving from the central region either towards the peripheral rim of the pars tensa or towards the manubrium. Furthermore, a local thickening was noticed in the antero-inferior quadrant of the membranes, and a strong linear correlation was observed between inferior-posterior length and mean thickness of the membrane. These features were combined into a single three-dimensional model to form an averaged representation of the human tympanic membrane. 3D reconstruction of the pathological tympanic membrane shows a structural atrophy with retraction pocket in the inferior portion of the pars tensa. The change of form at the pathological site of the membrane corresponds well with the decreased thickness values that can be measured there.

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.

Open access high-resolution 3D morphology models of cat, gerbil, rabbit, rat and human ossicular chains.

High-resolution 3D morphology models of cat, gerbil, rabbit, rat and human ossicular chains are presented. The models are based on high-resolution CT measurements. The resolution of the CT images, from which the models are segmented, varies from 5.6 to 33.5 µm. Models are freely available in different formats at our website (http://www.ua.ac.be/bimef/models) for research and educational purposes.

Anatomical boundary of the tympanic membrane pars flaccida of the Meriones unguiculatus (Mongolian gerbil).

The pars flaccida of the Meriones unguiculatus (Mongolian gerbil) was in previous studies shown to bulge almost spherically when pressurized, a behavior suggesting that it is suspended by a fixed circular boundary. The question arises whether this "functional" boundary is based on an underlying circular anatomical boundary, an important issue for modeling the middle-ear mechanics. In this article, the boundaries of the Mongolian gerbil pars flaccida were visualized in situ with otomicroscopy and in slides with light microscopy and by micro-CT radiology. For the major part of its circumference, the pars flaccida was found to be suspended by rigid bone, that is, the tympanic legs. The remaining boundary is made up of the terminal portion of the handle of the malleus and the soft tissue of the terminal arches. The attachment to these structures is simple and uncomplicated, and the geometry is regular and symmetric: deviating by only 3.5% from a perfect circular shape. The findings justify the use of a fixed circular boundary as a good approximation for the modeling of pars flaccida deformation in the Mongolian gerbil.

Realistic 3D computer model of the gerbil middle ear, featuring accurate morphology of bone and soft tissue structures.

In order to improve realism in middle ear (ME) finite-element modeling (FEM), comprehensive and precise morphological data are needed. To date, micro-scale X-ray computed tomography (µCT) recordings have been used as geometric input data for FEM models of the ME ossicles. Previously, attempts were made to obtain these data on ME soft tissue structures as well. However, due to low X-ray absorption of soft tissue, quality of these images is limited. Another popular approach is using histological sections as data for 3D models, delivering high in-plane resolution for the sections, but the technique is destructive in nature and registration of the sections is difficult. We combine data from high-resolution µCT recordings with data from high-resolution orthogonal-plane fluorescence optical-sectioning microscopy (OPFOS), both obtained on the same gerbil specimen. State-of-the-art µCT delivers high-resolution data on the 3D shape of ossicles and other ME bony structures, while the OPFOS setup generates data of unprecedented quality both on bone and soft tissue ME structures. Each of these techniques is tomographic and non-destructive and delivers sets of automatically aligned virtual sections. The datasets coming from different techniques need to be registered with respect to each other. By combining both datasets, we obtain a complete high-resolution morphological model of all functional components in the gerbil ME. The resulting 3D model can be readily imported in FEM software and is made freely available to the research community. In this paper, we discuss the methods used, present the resulting merged model, and discuss the morphological properties of the soft tissue structures, such as muscles and ligaments.

Evaluation of a model for studies on sequelae after acute otitis media in the Mongolian gerbil.

CONCLUSIONS: The model appears relevant for studies on sequelae after acute otitis media (AOM), and may be the seed of a new, chronic tympanic membrane perforation model in the gerbil. OBJECTIVES: To evaluate an experimental model for abortive otitis media and to assess the structural and functional changes of the tympanic membrane in the resolving phase. MATERIALS AND METHODS: The middle ears of 16 Mongolian gerbils were inoculated with type 6a Streptococcus pneumoniae. Half of the animals were treated with antibiotics on days 4-6, when otoscopy was performed as well. After 1, 2, 3 or 4 weeks the animals were sacrificed and their tympanic membranes were examined by otoscopy, dissection microscopy, light microscopy and moire interferometry. RESULTS: On days 4 and 6 AOM was produced in approximately 80% of the animals and perforations prevailed in approximately 30% at the study end points. Clinical signs of AOM and oedema of the tympanic membrane had already started to reduce after 1 week, and often resolved within 2 weeks. The mechanical stiffness of the tympanic membrane remained relatively unharmed in the non-perforated ears. The antibiotic treatment seemed to reduce the duration of oedema but not the perforation rate.

Design considerations in projection phase-shift moiré topography based on theoretical analysis of fringe formation.

Moiré topography is a well-established optical technique to measure the shape of three-dimensional surfaces, based on the geometric interference between an optical grid and its image deformed by an object surface. The technique produces fringes that represent contours of equal height, and from the recordings of several phase-shifted topograms surface height coordinates can be calculated. To perform these calculations, it is assumed that object height variation is small in comparison with the measurement setup dimensions, and this approximation leads to systematic errors in measurement accuracy. We present the mathematical description of the fringe formation process in projection moiré topography, and on the basis of these equations we establish the relation between setup geometry and upper limits of the systematic measurement errors. We derive the equations that determine design specifications needed to reduce the effects of approximations to be below the measurement resolution of the setup. It is shown that setup geometry should be adapted to the gray-scale measurement resolution of the imaging system. We show that, using an iterative correction from one fringe order to the next, measurement accuracy can be maintained over the entire object depth.