Jan A.N. Buytaert

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.

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.

Volume shrinkage of bone, brain and muscle tissue in sample preparation for micro-CT and light sheet fluorescence microscopy (LSFM)

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.

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.

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.

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.

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.

MicroFilament Analyzer, an image analysis tool for quantifying fibrillar orientation, reveals changes in microtubule organization during gravitropism.

Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high-throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi-stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin.

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. J. Morphol. 276:1025–1046, 2015.

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.