Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier-space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Feature extraction of chromosomes from 3-D confocal microscope images.

An investigation of local energy surface detection integrated with neural network techniques for image segmentation is presented, as applied in the feature extraction of chromosomes from image datasets obtained using an experimental confocal microscope. Use of the confocal microscope enables biologists to observe dividing cells (living or preserved) within a three-dimensional (3-D) volume, that can be visualised from multiple aspects, allowing for increased structural insight. The Nomarski differential interference contrast mode used for imaging translucent specimens, such as chromosomes, produces images not suitable for volume rendering. Segmentation of the chromosomes from this data is, thus, necessary. A neural network based on competitive learning, known as Kohonen's self-organizing feature map (SOFM) was used to perform segmentation, using a collection of statistics or features defining the image. Our past investigation showed that standard features such as the localized mean and variance of pixel intensities provided reasonable extraction of objects such as mitotic chromosomes, but surface detail was only moderately resolved. In this current work, a biologically inspired feature known as local energy is investigated as an alternative image statistic based on phase congruency in the image. This, along with different combinations of other image statistics, is applied in a SOFM, producing 3-D images exhibiting vast improvement in the level of detail and clearly isolating the chromosomes from the background. Index Terms-DIC, differential interference contrast, feature extraction, feature space, image segmentation, local energy, Morlet wavelet, phase congruency, self organizing feature map, SOFM.

High-aperture diffraction of a scalar, off-axis Gaussian beam

A scalar treatment for Gaussian beams offset from the optic axis and then focused by a high-numerical-aperture lens is presented. Such a theory is required for describing certain types of Doppler microscopes, i.e., when the measurement is simultaneously performed by more than a single beam axially offset and then focused by a lens. Analytic expressions for the intensity in the focal region of the high-aperture lens are derived. From these expressions we calculate the intensity in the focal region with parameters of beam size, beam offset, and the numerical aperture of the lens. The relative location and variation of the intensity around the focal region are discussed in detail. We show that for small-diameter Gaussian beams the Strehl ratio increases above unity as the beam is offset from the optic axis. This is explained by the increase in the effective numerical aperture of the offset beam compared with the one collinear with the optic axis. From examining the focal distribution, we conclude that it rotates for small beam size and that increasing beam diameter causes the focused distribution to rotate and shear, i.e., to distort. We also show that the distortion of the distribution increases with increasing numerical aperture.

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images.

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.

Minimum theoretical requirements for three-dimensional scanning-laser Doppler anemometry.

An investigation of the minimum number of intersecting beams that is required for laser Doppler anemometry (LDA) incorporating only a single detector is presented. We aim to provide decisive arguments for using four beams as the minimum requirement for complete three-dimensional velocity reconstruction even though three beams supply three velocity components. We derive expressions for the detected signals of the most general LDA system. From a matrix analysis of these expressions, we conclude that there is no physically realizable arrangement of three beams that results in complete three-dimensional velocity reconstruction and that four beams is the minimum number of beams required. We also determine the optimal arrangement of the four incident beams for unambiguous LDA and for best signal separation and immunity to minor optical alignment errors. To ascertain the velocity components, we scan the specimen in a precise manner relative to the point of focus of the beams, whereas some other researchers alter the frequency of the incident beams. The results obtained with these two methods are equivalent. However, scanning is mechanically simpler than frequency shifting and also allows for the formation of velocity images--images of the flow velocity over a region in two- or three-dimensional space. In particular, we examine systems that are limited by the common practice of using only a single high-numerical-aperture objective for both focusing and detection. We show that using high-numerical-aperture objectives results in the best signal differentiation and immunity to minor alignment errors.

Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMP.

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). We provide evidence here that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2- and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ.

A gene essential to brain growth and development maps to the distal arm of rat chromosome 12.

A recently discovered, spontaneous, autosomal recessive mutation in rats, flathead (fh), results in greatly reduced brain growth beginning in late fetal development. In this study we have mapped the fh mutation by determining the pattern of segregation of polymorphic microsatellite markers with respect to fh in 51 affected F2 offspring from a single interstrain intercross. Two markers on chromosome 12, D12Rat80 and D12Mgh6, cosegregated with the fh mutation in all 51 affected animals. The distribution of six additional markers in 40 informative meioses further localizes fh approximately 2 cM teleomeric to nos1. There are no known mutations in homologous regions of either mouse or human genomes that result in deficits in late neurodevelopment similar to that observed in fh/fh animals. The unique phenotype of fh/fh animals and the location of fh suggests the presence of a novel gene essential to normal brain development on the distal end of rat chromosome 12.

Fabrication and characterization of a planar gradient-index, plasma-enhanced chemical vapor deposition lens.

A thin, one-dimensional, gradient-index slab lens with a parabolic profile was designed and fabricated in fluorine-doped silica by use of plasma-enhanced chemical vapor deposition in a Helicon plasma reactor. The refractive-index profile of the fabricated lens was determined by the application of an inversion technique to the values of modal effective index measured with a prism coupler. The periodic refocusing property of the lens and the independence of the wavelength were measured with the fluorescence of a specially doped, thin polymer layer spin-coated onto the surface of the lens.

Confocal imaging of a stratified medium.

The confocal imaging of stratified media (for example, thin-film structures) is investigated. A simple model is introduced for the imaging of a single layer in order to explore the axial resolution attainable. A rigorous model is also described and compared with experimental results from thin surface films. A theoretical treatment of imaging of stratified media with a continuously varying refractive index is presented, and the inverse problem of reconstructing the refractive-index profile from a confocal image is discussed.

Choroid plexus cysts in the fetus: a benign anatomic variant or pathologic entity? Report of 41 cases and review of the literature.

During a 22-month period, 6288 women undergoing prenatal sonographic studies in the second and third trimesters were evaluated prospectively to determine the incidence of choroid plexus cysts in the fetus, to follow the natural course of these cysts in intrauterine life, and to determine the association of chromosomal and anatomic anomalies in these fetuses. We diagnosed choroid plexus cysts in 41 fetuses, an incidence of 0.65%. Unilateral and bilateral cysts were equally frequent, and in most cases diagnosed by 21 weeks' gestation. On follow-up scans, the cysts had completely disappeared by 23-24 weeks in 80% of the cases, and by 28 weeks in another 10%. Once resolved, the cysts did not recur, and a normal sonogram in the late second trimester predicted normal scans in late pregnancy and in the neonate. One fetus had a chromosomal abnormality (trisomy 18). Associated anatomic anomalies were detected in three fetuses, including the one with trisomy 18. We believe that in the great majority of cases, fetal choroid plexus cysts are benign transient variants of normal intracranial anatomy. It is, however, important to conduct a careful sonographic search for associated anomalies. Chromosomal studies are strongly recommended whenever associated anatomic abnormalities are detected and when the choroid plexus cysts are large, bilateral, and persistent beyond 20-22 weeks' gestation.

Sonographic estimation of fetal weight and Doppler analysis of umbilical artery velocimetry in the prediction of intrauterine growth retardation: a prospective study.

A prospective study was undertaken to examine whether Doppler velocimetry studies of the umbilical artery give a better or an earlier prediction of intrauterine growth retardation (IUGR) than do sonographic estimation of fetal weight. A total of 385 examinations were performed between 30 and 42 weeks of gestation on 168 patients who were at risk for IUGR. Forty-two (25%) of the patients delivered an infant with a birth weight below the tenth percentile. Although sensitivity was lower for the systolic to diastolic ratio (55%) of the umbilical artery than for the sonographic estimation of fetal weight (76%), the umbilical artery studies had a higher specificity (92% versus 80%) and predictive value of a positive test (73% versus 58%) when the last study to delivery interval was within 2 weeks. Furthermore, among 21 IUGR pregnancies with serial studies, the umbilical systolic to diastolic ratio was abnormal at a significantly earlier gestational age than when the sonographic estimate of fetal weight identified growth retardation. These findings indicate that sonographic biometry is a more sensitive technique for identifying IUGR but that the umbilical artery waveforms are a valuable adjunct for improving the diagnostic accuracy for the prenatal detection of IUGR. These data also provide suggestive evidence that umbilical artery velocimetry may be predictive of IUGR at an earlier gestational age than sonographic estimation of fetal weight.

Doppler umbilical velocimetry in the prediction of adverse outcome in pregnancies at risk for intrauterine growth retardation.

Perinatal indicators of fetal compromise were assessed according to the results of continuous-wave Doppler umbilical velocimetry for 172 patients at risk for intrauterine growth retardation (IUGR). Forty-three (25%) of the patients delivered an infant with a birth weight below the tenth percentile for gestational age. The last Doppler study before delivery was abnormal in 48.8% of the growth-retarded infants but in only 13.2% of the infants without evidence of IUGR. Furthermore, in the growth-retarded group, early delivery, reduced birth weight, decreased amniotic fluid at birth, admission to the neonatal intensive care unit, neonatal complications associated with IUGR, and a prolonged hospital stay were observed more frequently in those who had an abnormal ratio than in those with a normal ratio. The sensitivity of the systolic/diastolic ratio for an adverse perinatal outcome (operative delivery for fetal distress, neonatal morbidity associated with IUGR, and/or perinatal death) was significantly better for the infants with IUGR (66.7%) than for the infants without IUGR (27.8%; P less than .05). The predictive value of an abnormal ratio was also higher for the pregnancies complicated with IUGR (57.1%) than for those without IUGR (29.4%), but not to a statistically significant degree. These data suggest that Doppler umbilical velocimetry studies are valuable in identifying those growth-retarded fetuses at increased risk for an adverse perinatal outcome.