Quantitative Image Analysis Method Research Articles

Quantitative computed tomography analysis of local chemotherapy in liver tissue after radiofrequency ablation 1

Computed tomography (CT) was used to noninvasively monitor local drug pharmacokinetics from polymer implants in rat livers before and following radiofrequency ablation. Polymer matrixes containing carboplatin (a platinum-containing chemotherapeutic agent) were implanted into rat livers either immediately after radiofrequency ablation (n = 15) or without prior treatment (n = 15). The animals were divided into five subgroups (n = 3 per group) and subjected to a terminal CT scan at 6, 24, 48, 96, or 144 hours. Carboplatin concentration in tissue and within the implant matrix was correlated with CT intensity, and standard curves were produced for each environment. This correlation was used to evaluate the differences in drug transport properties between normal and ablated rat livers. A quantitative image analysis method was developed and used to evaluate the release rate and tissue distribution of carboplatin in normal and ablated liver tissue. The CT data were validated by previously reported atomic absorption spectroscopy measurement of implant and tissue drug levels. Correlation of carboplatin concentration and Hounsfield units results in a linear relationship with correlation coefficients (slopes) of 15 and 4 Hounsfield units/(mg/mL), for carboplatin in tissue and polymer, respectively. Noninvasive monitoring of local pharmacokinetics in normal and ablated tissues indicates that ablation before local carboplatin delivery increases the retention of carboplatin within the polymer matrix and drastically increases the drug retention in the ablated tissue volume (over 3-fold difference) resulting in a higher average dose to the surrounding tissue. At 1.6 mm from the implant boundary, carboplatin concentration is significantly higher in ablated tissue at 48, 96, and 144 hours (P <.05), and reaches 4.7 mg/mL in ablated tissue at 48 hours. In comparison, the concentration in normal liver at 1.6 mm reaches only 0.7 mg/mL at the same time point. The drug penetrates 3.1 mm in ablated liver compared with 2.3 mm in normal liver also at 48 hours. After 144 hours, the drug is still detected at 3.1 mm in ablated liver but not in normal liver. The differences are significant (P <.05) at both 48 and 144 hours. Correlation with chemical analysis suggests that CT data accurately predicts the drug pharmacokinetics in both ablated and normal livers. This work shows that X-ray CT imaging is a useful and promising technique for in vivo monitoring of the release kinetics of locally delivered radiopaque agents.

A Method for the Digital Image Analysis of Ceramic Grains Based on Shape Factor Segmentation

A very simple and robust method for ceramics grains quantitative image analysis is presented. Based on the use of optimal imaging conditions for reflective light microscopy of bulk samples, a digital image processing routine was developed for shading correction, noise suppressing and contours enhancement. Image analysis was done for grains selected according to their concavities, evaluated by perimeter ratio shape factor, to avoid consider the effects of breakouts and ghost boundaries due to ceramographic preparation limitations. As an example, the method was applied for two ceramics, to compare grain size and morphology distributions. In this case, most of artefacts introduced by ceramographic preparation could be discarded due to the use of perimeter ratio exclusion range.

Protein expression analysis using quantitative fluorescence image analysis on tissue microarray slides.

We have developed a tissue microarray (TMA)-based quantitative fluorescence image analysis (QFIA) method in which protein markers on TMA sections were labeled by immunofluorescence using tyramide signal amplification and a quantitative fluorescence detection system. Using this method, BRCA1 protein expression patterns were studied in the TMA sections of cell lines with known levels of BRCA1 expression and in a small group of human tissue samples obtained from sporadic epithelial ovarian cancers, their corresponding adjacent dysplastic fields, and distant non-tumor areas. We detected distinctive BRCA1 expression patterns in high-grade and low-grade sporadic epithelial ovarian cancers and their associated adjacent dysplastic fields. However, such patterns of expression could not be adequately detected by traditional immunohistochemical staining methods. TMA-QFIA provides a sensitive, automated, and quantitative measurement of protein expression on archived tissue and cell samples and will be a useful tool for protein-level molecular profiling analyses.

Desiccation of Late Quaternary inner shelf sediments: microfabric observations

Palaeo-desiccated crusts often form at the top of pre-Holocene shelf sediments during low sea-level stands resulting in significant changes in the mechanical properties. To assist in understanding these changes and the implications for Quaternary geology, the microfabric of sediment samples from the inner shelf off Hong Kong has been studied using scanning electron microscope techniques incorporating the latest methods of quantitative image analysis. These studies have been supported by petrographic observations and chemical analyses of the pore fluids. Holocene marine sediments usually have a honeycomb fabric while pre-Holocene marine sediments are more compact. In regions of high clay content, the palaeo-desiccated crust often possesses large planar voids filled with coarse silt-sized particles. This desiccated crust often contains relics of root traces filled with pyrite showing varying degrees of oxidation. The release of amorphous iron acts as a cementation agent and binds large silt/small sand grains giving a more compact fabric than that in neighbouring regions. Cementation in the pre-Holocene marine deposits creates strengthened open-fabric arrangements which may initially withstand moderate loads. Further evidence of desiccation comes from the presence of halite crystals in the pore fluid which has salt concentrations below that of normal sea water.

Effects of content and particle size of Si crystal on damping property of powder forged Al–Si alloy

The effects of the Si content and Si crystalline size on the damping properties of powder forged (PF) Al–Si alloys has been investigated using a fast Fourier transformation (FFT) analyser. A quantitative image analysis method was used successfully to determine the mean diameter and distribution of Si crystals in the aluminium alloy. The damping capacity greatly depends on the mean diameter of Si crystals. The value of δ for heat treated specimens with Si crystals of 2–5 μm is 4 to 25 times less than that for the as forged specimens with Si crystals of 1–5 μm. This indicates that the vibration is absorbed at the interface between the Si particles and the aluminium matrix by a mechanism of internal friction. Therefore, a fine distribution of Si crystals and/or elimination of crystal coarsening should contribute to improved damping.

Ａｌ‐Ｓｉ系粉末合金の減衰特性に及ぼすＳｉ含有量と粒子径の影響

Effects of the Si content and Si crystalline size on damping property of Powder Forged (P/F) Al-Si alloys were investigated by a Fast Fourier Transformation (FFT) analyzer. A quantitative image analysis method was successfully used to determine a mean diameter and distribution of Si crystals in the aluminum alloy. The damping capacity δ strongly depends on the mean diameter of Si crystals. δ of heat treated specimens with Si crystals of 2 to 5 micron meters is 4 to 25 times smaller than that of as-forged specimens with those of 1 to 5 micron meters. It indicates that the vibration occurred by knocking on the specimen is absorbed at boundary between Si particles and aluminum matrix. Therefore, fine distribution of Si crystals and/or elimination of crystal coarsening contribute to the good damping property.

Effects of an Oxidizing Agent (Hydrogen Peroxide) on the Glutathione System in Epidermal Cells of Allium cepa L. Investigated by Histochemical Staining

The glutathione system in epidermal cells of Allium cepa L. was measured by a quantitative image analysis method, using histochemical staining by monochlorobimane. Blockage of thiol groups with N-ethylmaleimide decreased fluorescence to a small rest fluorescence of maximum 5%. Pre-treatment with dithiothreitol increased fluorescence yield in all cells compared to the monochlorobimane treatment alone. These results correspond to the blockage or the reduction of the total pool of glutathione in the cells. After treatment with N-ethylmaleimide and subsequent incubation with dithiothreitol followed by labelling with bimane a low fluorescence yield was observed which correspond to the proportion of GSSG. Specimens that were incubated in H2O2 prior to the treatments described above exhibited a decrease in total glutathione and in reduced glutathione and an increase in the proportion of GSSG compared to the control.

Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

1. Biochemical and quantitative image analysis methods were used to investigate the anatomical basis for the previously described agonist-induced redistribution of calponin. 2. At 140 nm resolution, the quantitative distribution of calponin in resting cells was statistically indistinguishable from that of filament bundles containing alpha-smooth muscle actin and myosin, but was significantly different from that of filaments containing beta-non-muscle actin. Conversely, in stimulated cells, the distribution of calponin was not significantly different from that of beta-actin filaments in the subplasmalemmal cell cortex but was significantly different from the distribution of alpha-actin- and myosin-containing filamentous bundles. 3. The distribution of calponin significantly differed from that of the intermediate filament proteins vimentin and desmin as well as that of the dense body protein alpha-actinin either by ratio analysis of the subcellular distribution or by colocalization analysis. 4. The imaging results, although limited to 140 nm spatial resolution, suggested the hypothesis that the agonist-induced redistribution involves the binding of calponin to isoform-specific actin filaments. This hypothesis was tested by quantifying the relative affinity of calponin for purified alpha- and beta-actin. Light scattering measurements showed that calponin induces bundle formation with beta-actin more readily than alpha-actin, indicating that calponin may be preferentially sequestered by beta-actin under appropriate conditions. 5. These results are consistent with a model whereby agonist activation decreases calponin's binding to filaments, but the tighter binding to beta-actin filaments results in a spatial redistribution of calponin to the submembranous cortex.

Magnetic resonance imaging for the in vivo determination of body composition in animal science

Magnetic resonance imaging (MRI) is a diagnostic tool, originally developed for human medicine. It generates cross-sectional images of nearly all parts of the body with a high contrast between soft tissues. In animal science it has been utilized predominantly as an in vivo technique for the determination of body composition. The process of scanning live animals and especially the choice of the optimum measuring parameters is presented as well as methods of quantitative image analysis. Studies in pig and sheep indicate that quantification of lean and fat tissue by means of prediction equations is highly accurate. Coefficients of determination for the amount of lean and fat range between R 2 = 0.89 and R 2 = 0.97 for different weight groups. Direct measurements of breast muscle volume have been carried out in water fowl. Correlation between muscle volume derived from MR images and muscle weight due to dissection is abour r = 0.95. Based on the accurate prediction of lean and fat tissue and the volume measurement of single muscles MRI has been applied in pig, sheep and water fowl to analyse tissue und muscle growth during the fattening period. Influencing factors on growth, such as breed and sex, have been analysed by means of allometric functions. Furthermore in vivo imaging of morphological structures and pathological alterations is discussed.

Ferroelectric Domain Walls in BaTiO3: Fingerprints in XRPD Diagrams and Quantitative HRTEM Image Analysis

The structure of ferroelectric domain walls in BaTiO3 has been investigated through two complementary approaches, a global one by the fine analysis of X-ray diffraction patterns, the other essentially local via a quantitative image analysis method developed and applied to High Resolution Transmission Electron Microscopy images. These two original approaches converge towards a clear description of 90○ walls which are shown to be a 4–6 nm wide region where the crystallographic discontinuity is accommodated by irregular atomic displacements. The results given here demonstrate that the usual structural theoretical description of walls commonly accepted for energy calculations are far too simplistic. The two underlying methodologies which have been developed to carry out these approaches can possibly be applied to other ferroelectrics, but without any doubt to other systems where twins or coherent interfaces are expected.

A quantitative image analysis method for characterizing mixtures of granular materials

Automated image analysis is used to characterize the structure of undisturbed granular mixtures. These mixtures, produced from small-scale mixing experiments, are preserved using solidification techniques, sliced, and scanned using a video camera to produce a digital image. Image analysis is used to determine the mean, mode, standard deviation, variance, and skewness of local composition for a large number of locations in the mixtures, providing an effective method for performing a detailed quantitative characterization of the mixture structure. The digital data are then used to simulate random sampling from a powder blender. Sampling parameters, such as number of samples, sample size, and location of the samples, are varied to assess their effect on the descriptive statistics. For partially mixed systems such as those examined here, results depend strongly on the number of samples used in the analysis, but they are largely independent of sample size. The solidification techniques, image analysis methods, and numerical algorithms for quantifying features of the partially mixed structures are described in detail.

Quantitative Description of the Evolution in the Geometry of Grains in a Pure α-Fe

Grains in polycrystals differ in their size and shape. The geometry of grains in a given polycrystal is described by the distribution functions of grain size and shape. These functions can be experimentally studied using the methods of quantitative stereology and image analysis. The geometry of grains is related to the physical propeties of the grain boundaries and vice versa. The distribution function of the geometrical features of grains can help in the assessment of the physical properties of the grain boundaries. Distribution functions of grains size and shape were investigated in an UHP iron produced at EMSE and in a commercially available Armco iron. Populations of grains in the specimens studied were charaterized in terms of mean grain volume, E(V), grain section area, E(A) and the grain size diversity ratio. The data obtained in the study provide a quantitative description of the evolution in the geometry of grains during recrystallization and grain growth It was found that there are systematic differences in the geometrical features of grains in UHP iron and Armco iron. The possible contribution of these differences to the disparity in the properties of the UHP materials is discussed.

Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis

Although filamentous microorganisms are widely used in industrial fermentation processes, their growth and differentiation are not yet fully understood, because their biomass is structured, and therefore difficult to describe and to quantify. This lack of appropriate tools can hinder the optimization and control of the fermentation. A quantitative image analysis method was therefore developed for characterizing the physiology of the penicillin-producing mold Penicillium chrysogenum. This method is based on a differential staining procedure showing six physiological states: growing material, three differentiated states characterized by an increasing granulation, a highly vacuolized state, and dead segments having lost their cytoplasm. The image analysis software, with versions written for monochrome and color images, consisted of a semiautomatic binary mask computation step and a fully automatic segmentation step based on a fuzzy classification.

A quantitative image analysis method for the determination of cocontinuity in polymer blends

A new morphological parameter, ‘CoContinuity’, has been developed and implemented for quantitative measurement of morphology in cocontinuous blends of polymers. The basis of the CoContinuity is the extent to which the phases of a polymer blend mutually surround each other. A secondary parameter, ‘CoContinuity Balance’, is also presented to describe quantitatively the relative contribution of each phase to the CoContinuity. Both of these functions are dimensionless and scale-invariant, thus allowing objective comparisons of dissimilar systems. An example is given where the functions faithfully describe the evolution of a polycarbonate/poly(styrene- co-acrylonitrile) (PC/SAN) blend of varying phase ratio as the system progresses from discrete domains of the SAN phase in a matrix of PC, through a cocontinuous morphology of SAN and PC, and finally ends up as a mixture of discrete domains of PC in a matrix of SAN.

A quantitative image analysis method for the determination of cocontinuity in polymer blends

Two new morphological measurements based on digital image analysis, CoContinuity and CoContinuity Balance, have been developed and implemented for quantitative measurement of morphology in polymer blends. The morphology of polymer blends varies with phase ratio, composition and processing. A typical morphological evolution for increasing phase ratio of polymer A to polymer B starts with discrete domains of A in a matrix of B (A/B &lt; 1), moves through a cocontinuous distribution of A and B (A/B ≈ 1) and finishes with discrete domains of B in a matrix of A (A/B &gt; 1). For low phase ratios, A is often seen as solid convex particles embedded in the continuous B phase. As the ratio increases, A domains begin to evolve into irregular shapes, though still recognizable as separate domains. Further increase in the phase ratio leads to A domains which extend into and surround the B phase while the B phase simultaneously extends into and surrounds the A phase.

Formula omitted] differentially affects LH release and LHRH mRNA levels in estrogen-treated ovariectomized control and androgen-sterilized rats

Excitatory amino acids such as N- methyl- d,l-aspartic acid (NMDA) are thought to play an important role in the regulation of gonadotropin secretion. NMDA induces significant increases in plasma LH in a variety of animal models and these effects occur by activation of neural processes involved in excitation of LHRH neurons rather than by a direct action on the pituitary gland. We have taken advantage of this information to study the effects of NMDA on LH release and on changes in levels of LHRH mRNA in single neurons of adult rats treated neonatally with a high dosage of androgen. While iv NMDA evoked an increase in plasma LH in estrogen-treated ovariectomized control and androgen-sterilized rats (ASR), significantly less LH was released in ASR. LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT), the rostral (r), medial (m) and caudal (c) preoptic (POA) regions were quantitated using in situ hybridization histochemistry and quantitative image analysis methods. LHRH mRNA levels in untreated controls and ASR did not differ in any of the brain regions examined. Within 1 h after NMDA, LHRH mRNA had increased significantly in OVLT and rPOA but not in mPOA and cPOA neurons of control rats and these mRNA levels remained elevated for 4 h. In contrast, NMDA treatment of ASR did not affect basal levels of LHRH mRNA in any region of the rostral hypothalamus. These observations suggest that neonatal androgen treatment of female rats either directly or indirectly affects the responsiveness of LHRH neurons to NMDA. As a consequence, LH release is attenuated and the ability of these neurons to respond to NMDA by an increase in LHRH mRNA is lost.

Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels Increase in A1 but not Locus Ceruleus Noradrenergic Neurons in Proestrous Rats but not in Diestrous or Androgen‐Sterilized Animals

Norepinephrine (NE) turnovers (an index of secretion) increase in the hypothalamus of proestrous rats concomitant with luteinizing hormone surges, whereas, neither of these events are observed in diestrous nor in androgen-sterilized rats. Increased hypothalamic NE release may occur as a consequence of the withdrawal of local inhibitory γ-aminobutyric acid and opiate controls on specific presynaptic NE terminals and/or as a result of an increase in activity within noradrenergic neurons. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the synthesis of NE and our earlier studies revealed that increases in TH mRNA in A1 and locus ceruleus (LC) neurons can serve as an index of increased activity within these cells. In the present study, we evaluated whether TH message levels change in A1 and LC neurons prior to and during the hours when luteinizing hormone surges and increased NE turnovers are observed. As controls, TH mRNA levels in A1 and LC neurons were evaluated at the same hours of day in diestrous day 2 and in androgen-sterilized rats. In situ hybridization histochemistry and quantitative image analysis methods were used to measure changes in TH mRNA levels. Luteinizing hormone surges in proestrous rats began at 1500 h, peaked between 1600 and 1700 h and declined, thereafter, to 2000 h. In contrast, plasma luteinizing hormone remained basal throughout the day in diestrous and androgen-sterilized rats. While A1 neuronal TH mRNA levels did not differ in the three groups of rats during the morning (0930 to 1030 h), these message levels were significantly elevated in proestrous rats during the afternoon (1645 to 1715 h) and remained high at 2000 to 2030 h. In contrast, no changes in TH mRNA levels were observed in A1 neurons throughout the afternoon in diestrous animals or androgen-sterilized rats. TH mRNA levels in the LC did not differ in the three groups of rats and they remained unchanged throughout the afternoon hours we examined. From these observations we conclude that concomitant with afternoon proestrous luteinizing hormone surges and the accompanying increase in hypothalamic NE secretion, there is an increase in activity within A1 but not LC neurons. These data suggest that the proestrous increase in hypothalamic NE turnover we previously observed is not due solely to withdrawal of local inhibitory controls of presynaptic NE release but it also involves an increase in activity within A1 but not LC neurons.

Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods

These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear.

Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons

We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after beginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral A1 or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur.

Postnatal development of the noradrenergic projection from locus coeruleus to the olfactory bulb in the rat

Norepinephrine (NE) may play a role in the developing brain by modulating synaptic plasticity during critical periods of circuit formation (Kasamatsu and Pettigrew, 1976; 1979; Bear and Singer, 1986). In the olfactory bulb, NE input from the locus coeruleus (LC) appears to be necessary for the newborn rat to form a learned odor preference (Sullivan and Leon, 1986; Wilson and Leon, 1988; Sullivan et al., 1989). However, little is known about the development of NE innervation of the olfactory bulb. Thus, it is not clear how the maturation of the LC projection to the bulb correlates with the formation of olfactory bulb circuits during the period when NE modulates early olfactory learning. In this study, the postnatal development of the NE input from the LC to the main and accessory bulbs was characterized with tract tracing, immunocytochemistry, and quantitative image analysis methods. By birth there is already a substantial input to the olfactory bulb from the LC; as many as 200 LC neurons can be retrogradely labelled with wheatgerm agglutinin-horseradish peroxidase injection in the olfactory bulb. This compares with an estimated 400-600 neurons labelled by similar procedures in adult rats (Shipley et al., 1985). In order to study the development of NE fibers innervating the olfactory bulb, immunocytochemistry with antibodies to dopamine-beta-hydroxylase was employed. Image analysis was used to facilitate visualization and to quantitate the development of fiber densities. At birth, immunocytochemically labelled NE fibers were identified in all layers of the main and accessory olfactory bulb. The innervation was strongly preferential for infraglomerular layers at all stages of postnatal development. The fibers were densest in the internal plexiform and granule cell layers, less dense in the external plexiform layer, and sparse in the glomerular layer. The density of the fibers increased during development. There were no significant shifts in the relative distribution of the fibers in different layers of the bulb during development. This consistent laminar innervation by NE fibers suggests that if these fibers have a developmental role, their influence is probably limited to neuronal elements in inframitral cell layers.