Joris Dirckx

Refractive index of tissue measured with confocal microscopy

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382+/-0.004, at 592 nm.

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.

Volume displacement of the gerbil eardrum pars flaccida as a function of middle ear pressure

The pars flaccida (PF) is a small region of the eardrum, with elasticity and histology completely different from the rest of the membrane, which has often been attributed a pressure regulating function for the middle ear (ME). In this paper, the volume displacement of the PF as a function of ME pressure is discussed. The deformation of the PF was measured in vitro in five Mongolian gerbil ears, by means of an opto-electronic moiré interferometer. Volume displacement was determined at small intervals in three sequential pressure cycles, in the range of +/- 0.4 kPa, +/- 2 kPa, and again +/- 0.4 kPa. The displacement was found to be a highly non-linear function of pressure, with a strong increase up to 0.4 kPa ME over- or underpressure and remaining nearly unchanged for pressures beyond 0.4 kPa. In all animals, maximal volume displacement was less than 0.5 microl, or 0.2% of total ME air volume. Clear hysteresis was found between the deformations at the same pressure level in the increasing and decreasing parts of the pressure cycles. Membrane behavior in the first 0.4 kPa pressure cycle was significantly different from that in the second 0.4 kPa cycle, which followed the 2 kPa pressure cycle. The results indicate that the ME pressure change regulation function of the PF is limited to very small pressure changes of a few hundred Pa around ambient pressure, and that larger ME pressures cause at least short-term changes in the membranes behavior.

Phase shift method based on object translation for full field automatic 3-D surface reconstruction from moire topograms

A new method fur full field automatic 3-D surface reconstruction is proposed which makes use of multiple contourograms shifted in phase by object translation. The method is demonstrated for shadow moire topography. It is shown that surface reconstruction can be done fast and with a resolution at least 10 times higher than the fringe distance of the measuring setup. Convexity and concavity of the surface are automatically determined. Also shown is the possibility of measuring irregular surfaces with very sudden height jumps.

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.

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.

Human tympanic membrane deformation under static pressure

The effect of static pressures in the range of plus and minus 1.6 kPa on the shape of tympanic membrane is measured using a non-contacting optical technique on a fresh human temporal bone. Full field data of the deformation are presented as well as cross-sections along two major directions. Strong asymmetry between medial and lateral movements is demonstrated. The displacement of the umbo is compared to other work. The rotation angle of the manubrium in function of pressure is calculated and also compared to other work. It is demonstrated that the rotation angels can not account for the measured movement of the umbo, which leads to the conclusion that for static high pressure levels the classical hypothesis of rotation around a fixed axis has to be abandoned. The comparison with data of TM displacement under dynamic stimuli is discussed.

Displacement of the gerbil tympanic membrane under static pressure variations measured with a real-time differential moire interferometer

It is thought that chronic middle ear disease ultimately causes changes in the stiffness and elasticity of the tympanic membrane, but it is unknown whether such changes occur early in the course of the disease. In order to analyze mechanical changes in different parts of the tympanic membrane, a full field moire interferometry technique was utilized to measure the shape and real-time displacement in response to positive and negative pressure gradients applied across the tympanic membrane. The measurements were performed on fresh isolated temporal bones from the Mongolian gerbil. In order to gain sufficient visual access to the pars tensa for the moire measurements, the tympanic bulla was opened, the tensor tympani muscle and the incudo-stapedial joint were cut, and part of the medial wall of the tympanic cavity was removed. The malleus and incus and their ligaments were kept intact. The specimens were kept continuously humidified with an evaporator or in a humid chamber, since otherwise the tympanic membrane dries out in a few minutes when its medial surface is exposed. This desiccation reduces the elasticity and cause shrinkage which results in a reduction of the height of the cone constituted by the pars tensa. Profiles of the tympanic membrane at rest and under different pressure conditions were extracted from the moire interferograms. The tympanic membrane and ossicular complex exhibit a hysteresis effect as differences in the displacement patterns under identical pressure gradients during the loading and the unloading phase; a residual displacement of the pars tensa was for instance seen after the pressure gradient across the tympanic membrane was eliminated.

Shape and derived geometrical parameters of the adult, human tympanic membrane measured with a phase-shift moiré interferometer

The shape of the tympanic membrane is fairly complex and seems to be of significant importance in the coupling of the acoustic sound pressure in the external ear canal to the motion of the middle ear ossicles. A moiré shift interferometer was used to measure with great precision the shape of the external surface of human tympanic membrane. The dense matrix of z(x,y) values thus obtained is used to calculate different geometrical parameters (area, curvature, ...). We show further how the same data can be used to specify exactly the shape of the tympanic membrane in a mathematical finite-element model of the middle ear.

Effect of middle ear components on eardrum quasi-static deformation

Eardrum deformation induced by quasi-static middle ear pressure was studied at progressive stages of dissection of gerbil temporal bones. With our high resolution moiré interferometer we recorded the shape and deformation of the eardrum along a line perpendicular to the manubrium and through the umbo, at different middle ear pressures. The deformation was measured from the medial side, after serially removing the cochlea, removing the stapes, cutting the tensor tympani, exposing the incudo-mallear joint, and cutting the anterior bony process which connects the malleus to the tympanic bone. The mean displacement as a function of pressure was also determined at all stages of dissection. Removing the cochlea and stapes, and cutting tensor tympani has no effect on static eardrum deformation. Exposing the incudo-mallear joint increases eardrum movement, and cutting the anterior bony connection between malleus and temporal bone strongly changes eardrum rest position and further increases its displacement.