Two methods are especially suited for tomographic imaging with histological detail of macroscopic samples that consist of multiple tissue types (bone, muscle, nerve or fat): Light sheet (based) fluorescence microscopy (LSFM) and micro-computed tomography (micro-CT). Micro-CT requires staining with heavy chemical elements (and thus fixation and sometimes dehydration) in order to make soft tissue imageable when measured alongside denser structures. LSMF requires fixation, decalcification, dehydration, clearing and staining with a fluorescent dye. The specimen preparation of both imaging methods is prone to shrinkage, which is often not mentioned, let alone quantified. In this paper the presence and degree of shrinkage are quantitatively identified for the selected preparation methods/stains. LSFM delivers a volume shrinkage of 17% for bone, 56% for muscle and 62% for brain tissue. The three most popular micro-CT stains (phosphotungstic acid, iodine with potassium iodide, and iodine in absolute ethanol) deliver a volume shrinkage ranging from 10 to 56% for muscle and 27-66% for brain, while bone does not shrink in micro-CT preparation.

The full-field thickness distribution, three-dimensional surface model and general morphological data of six human tympanic membranes are presented. Crosssectional 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 interspecimen 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.

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.

In order to improve realism in middle ear (ME) finiteelement 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 opticalsectioning 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.

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.