Imaging and characterizing cells using tomography.

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft X-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft X-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft X-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells.

Pre-spermiogenic initiation of flagellar growth and correlative ultrastructural observations on nuage, nuclear and mitochondrial developmental morphology in the zebrafish Danio rerio.

The microstructural and ultrastructural changes of germ cells during spermatogenesis of zebrafish (Danio rerio) were examined using light microscopy (LM) and transmission electron microscopy (TEM). Generally the process of spermatogenesis in zebrafish is similar to that of other teleosts, however, here we describe some peculiar features of zebrafish spermatogenic cells which have a limited report in this species. (1) The basic events of spermiogenesis are asynchronous, location of flagellum finished in initial stage, while chromatin condensation sharply occurred in intermediate stage and elimination of excess cytoplasm mainly taken place in final stages. (2) Surprisingly, the cilia or initial flagellae are created in spermatocytes, approach toward the nucleus of early stage spermatids, and then the centrioles depress into nuclear fossa and change their orientation to each other from right angle to obtuse angle about 125°. (3) During spermatogenesis, the chromatin compaction performs in a distinctive pattern, condensed heterogeneously from granular into chromatin clumps with central electron-lucent areas, round or long, which diminished to small nuclear vacuoles in spermatozoa. This finding demonstrates the origin of nuclear vacuoles in zebrafish spermatozoa for the first time. (4) Nuages are observed in both spermatogonia and spermatocytes. They are connected with the mitochondria and nuclear membrane, and are even located in the perinuclear spaces of spermatogonia nuclei. (5) Mitochondrial morphology and distribution shows diversity in different germ cells. The condensed mitochondria appear in pachytene spermatocytes, and mitochondria including membrane conglomerate exist in both spermatocytes and spermatids. This study was undertaken in order to disclose specific spermatogenic cells features in zebrafish that could be helpful for understanding the correlative function in this model species.

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes.

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition. RESULTS: In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents. CONCLUSIONS: The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.

Automated microscopic image analysis for leukocytes identification: a survey.

Automatic quantification and classification of leukocytes in microscopic images are of paramount importance in the perspective of disease identification, its progress and drugs development. Extracting numerical values of leukocytes from microscopic images of blood or tissue sections represents a tricky challenge. Research efforts in quantification of these cells include normalization of images, segmentation of its nuclei and cytoplasm followed by their classification. However, there are several related problems viz., coarse background, overlapped nuclei, conversion of 3-D nuclei into 2-D nuclei etc. In this review, we have categorized, evaluated, and discussed recently developed methods for leukocyte identification. After reviewing these methods and finding their constraints, a future research perspective has been presented. Further, the challenges faced by the pathologists with respect to these problems are also discussed.

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta, Nereidae).

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a "9+2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning - free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.

Imaging the inside of a tumour: a review of radionuclide imaging and theranostics targeting intracellular epitopes.

Molecular imaging of tumour tissue focusses mainly on extracellular epitopes such as tumour angiogenesis or signal transduction receptors expressed on the cell membrane. However, most biological processes that define tumour phenotype occur within the cell. In this mini-review, an overview is given of the various techniques to interrogate intracellular events using molecular imaging with radiolabelled compounds. Additionally, similar targeting techniques can be employed for radionuclide therapy using Auger electron emitters, and recent advances in Auger electron therapy are discussed.

Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation.

PURPOSE: The authors' aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF). METHODS: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10-100 keV), an x-ray beam (100 kVp), and (125)I and (103)Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared. RESULTS: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2) using 30 nm AuNPs, would require 5.1 ± 0.2, 9 ± 1, 10 ± 1, 10 ± 1 mg Au/g tissue in the nucleus, in the cytoplasm, on the cell surface, or in the extracellular space, respectively. Using 50 nm AuNPs, the required amount decreases to 3.1 ± 0.3, 8 ± 1, 9 ± 1, 9 ± 1 mg Au/g tissue, respectively. CONCLUSIONS: NDEF is a new metric that can predict the radiation enhancement of AuNPs for various experimental conditions. Cell model, the subcellular location and size of AuNPs, and the number of AuNPs per cell, as well as the x-ray photon energy all have effects on NDEFs. Larger AuNPs in the nucleus of cluster cells exposed to x-rays of 15 or 40 keV maximize NDEFs.

Computing the length of the shortest telomere in the nucleus.

The telomere length can either be shortened or elongated by an enzyme called telomerase after each cell division. Interestingly, the shortest telomere is involved in controlling the ability of a cell to divide. Yet, its dynamics remains elusive. We present here a stochastic approach where we model this dynamics using a Markov jump process. We solve the forward Fokker-Planck equation to obtain the steady state distribution and the statistical moments of telomere lengths. We focus specifically on the shortest one and we estimate its length difference with the second shortest telomere. After extracting key parameters such as elongation and shortening dynamics from experimental data, we compute the length of telomeres in yeast and obtain as a possible prediction the minimum concentration of telomerase required to ensure a proper cell division.

The influence of spatial variation in chromatin density determined by X-ray tomograms on the time to find DNA binding sites.

In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process.

Organization of organelles within hyphae of Ashbya gossypii revealed by electron tomography.

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.

3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.

Optimal ultraviolet wavelength for in vivo photoacoustic imaging of cell nuclei.

In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.

Plasmodium induced by SU6656, an Src family kinase inhibitor, is accompanied by a contractile ring defect.

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei.

Fusion failure of dense-cored proacrosomal vesicles in an inducible mouse model of male infertility.

The acrosome is a specialized secretory vesicle located in the head of spermatozoa and has an essential role during fertilization. This organelle and the sperm nucleus have aberrant morphologies in forms of male infertility in humans (teratozoospermia), often associated with poor motility (asthenoteratozoospermia). To further our understanding of the aetiology of these conditions, we have performed a pathological investigation of a model of asthenoteratozoospermia that can be induced in mice by N-butyldeoxynojirimycin (NB-DNJ). We have found that, in mice treated with NB-DNJ, instead of an acrosome forming over the round spermatid nucleus, multivesicular bodies (MVB) accumulate in the vicinity of this nucleus. Electron microscopy has revealed that proacrosomic vesicles or granules (PAG) secreted during the Golgi phase of spermiogenesis do not fuse together to form an acrosomic vesicle, but rather attach transiently to the spermatid nucleus. Immunocytochemistry has shown that acrosomal membrane proteins and cytosolic acrosome-associated proteins are redirected to MVB in affected testes, whereas glycoproteins originating in the dense core of the PAG are degraded. Thus, the major effect of NB-DNJ is to inhibit membrane fusion of Golgi-derived secretory vesicles destined for acrosome formation, raising the possibility that these vesicles are critically affected in forms of (astheno)teratozoospermia.

Effects of cell spatial organization and size distribution on ultrasound backscattering.

In ultrasound tissue characterization dealing with cellular aggregates (such as tumors), it can be hypothesized that cell microstructure and spatial distribution dominate the backscatter signal. Effects of spatial organization and size distribution of nuclei in cell aggregates on ultrasound backscatter are examined in this work using 2-D computer simulations. The nuclei embedded in cytoplasm were assumed to be weak scatterers of incident ultrasound waves, and therefore multiple scattering could be neglected. The fluid sphere model was employed to obtain the scattering amplitude for each nucleus and the backscatter echo was generated by summing scattered signals originating from many nuclei. A Monte Carlo algorithm was implemented to generate realizations of cell aggregates. It was found that the integrated backscattering coefficient (IBSC) computed between 10 and 30 MHz increased by about 27 dB for a spatially random distribution of mono-disperse nuclei (radius = 4.5 µm) compared with that of a sample of periodically positioned mono-disperse nuclei. The IBSC also increased by nearly 7 dB (between 10 and 30 MHz) for a spatially random distribution of poly-disperse nuclei (mean radius ± SD = 4.5 ± 1.54 µm) compared with that of a spatially random distribution of mono-disperse nuclei. Two different Gaussian pulses with center frequencies 5 and 25 MHz were employed to study the backscatter envelope statistics. An 80% bandwidth was chosen for each case with approximately 0.32 mm as the full-width at half-maximum (FWHM) for the first pulse and 0.06 mm for the second. The incident beam was approximated as a Gaussian beam (FWHM = 2.11 and 1.05 mm for those pulses, respectively). The backscatter signal envelope histograms generally followed the Rayleigh distribution for mono-disperse and poly-disperse samples. However, for samples with partially ordered nuclei, if the irradiating pulse contained a frequency for which ultrasound wavelength and scatter periodicity became comparable (d ~ λ/2), then the histograms were better fitted by the Nakagami distribution. This study suggests that the shape of an envelope histogram depends upon the periodicity in the spatial organization of scatterers and bandwidth of the ultrasound pulse.

Ultrasonic backscatter coefficient quantitative estimates from Chinese hamster ovary cell pellet biophantoms.

A cell pellet biophantom technique is introduced, and applied to the ultrasonic backscatter coefficient (BSC) estimate using Chinese hamster ovary (CHO) cells. Also introduced is a concentric sphere scattering model because of its geometrical similarities to cells with a nucleus. BSC comparisons were made between the concentric sphere model and other well-understood models for mathematical verification purposes. BSC estimates from CHO cell pellet biophantoms of known number density were performed with 40 and 80 MHz focused transducers (overall bandwidth: 26-105 MHz). These biophantoms were histologically processed and then evaluated for cell viability. Cell pellet BSC estimates were in agreement with the concentric sphere model. Fitting the model to the BSC data yielded quantitative values for the outer sphere and inner sphere. The radius of the cell model was 6.8 ± 0.7 µm; the impedance of the cytoplasm model was 1.63 ± 0.03 Mrayl and the impedance of the nuclear model was 1.55 ± 0.09 Mrayl. The concentric sphere model appears as a new tool for providing quantitative information on cell structures and will tend to have a fundamental role in the classification of biological tissues.

Achieving the way for automated segmentation of nuclei in cancer tissue images through morphology-based approach: a quantitative evaluation.

In this paper we address the problem of nuclear segmentation in cancer tissue images, that is critical for specific protein activity quantification and for cancer diagnosis and therapy. We present a fully automated morphology-based technique able to perform accurate nuclear segmentations in images with heterogeneous staining and multiple tissue layers and we compare it with an alternate semi-automated method based on a well established segmentation approach, namely active contours. We discuss active contours' limitations in the segmentation of immunohistochemical images and we demonstrate and motivate through extensive experiments the better accuracy of our fully automated approach compared to various active contours implementations.

Clinical utility of squamous and transitional nuclear structure alterations induced by Schistosoma haematobium in chronically infected adults with bladder damage verified by ultrasound in Ghana.

OBJECTIVE: To evaluate the clinical utility of quantitative nuclear morphometry--i.e., alteration in nuclear size/shape, DNA content and chromatin structure-of intact cells obtained from the sediment of urine specimens collected from people living in an area highly endemic for Schistosoma haematobium in Ghana. STUDY DESIGN: Digital images of Feulgen-DNA-stained squamous cell (SC) and transitional cell (TC) urothelial nuclei were captured using the AutoCyte imaging system, and nuclear morphometric descriptors (NMDs) were calculated. A total of 3,495 and 4,523 SC and TC nuclei from normal bladder ultrasound subjects (n =21) and 3,465 and 3,064 SC and TC nuclei from severely abnormal bladder ultrasound subjects (n = 20) were captured. RESULTS: Univariate logistic regression analyses of pooled SC and TC nuclei training sets showed that 27/40 NMDs and 24/40 NMDs were univariately significant for differentiating between SCs and TCs of subjects with normal and severely abnormal bladder ultrasound. Multivariate models constructed using NMDs with > or = 50% inclusion frequency yielded AUC-ROCs of 75.23% and 74.42% in the SC training and validation, and 69.90% and 66.70% for TC training and validation. Further, a squamous cell patient-specific model predicted severe bladder damage with an AUC-ROC of 86.90%, yielding the sensitivity, specificity and accuracy of 85.00%, 76.19% and 80.49%, respectively. CONCLUSION: Quantitative nuclear structure alterations can be used to make a noninvasive assessment of cytologic changes observed in both SC and TC bladder epithelia due to S haematobium infection.

Colour-texture based image analysis method for assessing the hormone receptors status in breast tissue sections.

Hormone receptors have been used in prognosis of breast carcinomas and their positive status is of clinical value in hormonal therapy. Determination of this status is based on the subjective visual inspection of the stained nuclei in the specimens. The aim of this study was the assessment of the estrogen receptor's (ER) positive status of breast carcinomas, by means of colour-texture based image analysis methodology. Twenty two cases of immunohistochemically (IHC) stained breast biopsies were initially assessed by a histopathologist for ER positive status, following a clinical scoring protocol. Custom-designed image analysis software was developed for automatically assessing the ER positive status, employing colour textural features and the k-Nearest Neighbor weighted votes classification algorithm. Computer-based image analysis system resulted in 86.4% overall accuracy and in 0.875 Kendall's coefficient of concordance (p<0.001), ranking correctly 19/22 cases. Colour-texture analysis of IHC stained specimens might have an impact in the quantitative assessment of ER status.

[Nuclear neuroimaging of dopamine transporter in Parkinsonism--role in routine clinical practice].

Idiopathic Parkinson's disease (IPD) is a neurodegenerative condition characterized pathologically by the degeneration of dopaminergic neuron in the substantia nigra and production of intracytoplasmic inclusion bodies (Lewy body) in the retained neurons. Clinically, the disease is characterized by the presence of tremor, rigidity and bradykinesia. These clinical features also occur in other neurodegenerative diseases and by dopamine receptor antagonist drugs. Brain SPECT imaging of the dopamine transporter (DAT) with specific radioligands is a sensitive method for examining the integrity of the presynaptic dopaminergic system. However, with this main clinical application it is hard to diagnose patients with mild, incomplete, or uncertain Parkinsonism. The ligands belong to a group of compounds derived from cocaine that bind to the dopamine transporter and include â-CIT, IPT, TRODAT-1, FP-CIT tagged with either Iod-123 or Technetium-99m radioisotopes. DAT imaging is abnormal even in the earliest clinical presentation of IPD but a normal scan suggests an alternative diagnosis such as essential tremor, vascular Parkinsonism, drug-induced Parkinsonism, or psychogenic Parkinsonism.