Cellular Morphometry Research Articles

Perfil hematológico e bioquímica sérica de catetos (Pecari tajacu Linnaeus, 1758) no semiárido do nordeste brasileiro

ABSTRACT Hematological and biochemical analyses are paramount in assessing the health of different populations and their habitats, and knowledge on the specific values of each species is necessary, due to influence of several factors. In this context, this study aimed to determine the hematological and biochemical parameters of collared peccaries raised in captivity in the semi-arid Brazilian northeast associated to morphometric blood cell analyses. A total of 30 adult animals were investigated, divided into two groups (15 females and 15 males), averaging 20 kg and between 2 and 2.5 years old. Blood and serum samples were collected in the morning for hematological, serum biochemistry and cellular morphometry analyses. Data normality was determined by the Shapiro-Wilk test, and homoscedasticity, by Levene’s test. Concerning statistical differences between sexes, parametric data were analyzed by the t test for independent measurements and non-parametric data, by the Mann-Whitney test. Sex did not influence most of the studied variables, except for calcium, total proteins and urea and relative eosinophil counts. The environment and containment manner can influence collared peccary hematological and biochemical parameters, and blood cell morphometry and morphology data are similar to those of domestic cattle and carnivores.

Scattered-light-sheet microscopy with sub-cellular resolving power.

Since its first demonstration over 100 years ago, scattering-based light-sheet microscopy has recently re-emerged as a key modality in label-free tissue imaging and cellular morphometry; however, scattering-based light-sheet imaging with subcellular resolution remains an unmet target. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time-averaged pseudo-thermalized light-sheet illumination. While this approach increased the lateral dimensions of the illumination sheet, we achieved subcellular resolving power after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity, no staining, and ultralow irradiance levels. Overall, we expect this scattering-based light-sheet microscopy approach will advance single, live cell imaging by conferring low-irradiance and label-free operation towards eradicating phototoxicity.

FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial-to-mesenchymal transition induced by TGF-β in prostate cancer cells.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-β) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-β treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm-1 and 3100-2800 cm-1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm-1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-β treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities.

Abstract 1926: Machine learning-enhanced image and spatial analytic pipelines for imaging mass cytometry applied to the TRACERx non-small cell lung cancer study

Abstract Introduction: High dimensional imaging approaches such as imaging mass cytometry (IMC) are becoming widely used in cancer research. Such methods allow simultaneous observation of many cell types and their functional states and can provide valuable spatial information on cancer disease states when applied to clinical tissue samples. For example, in-situ immune and tumor cell interactions can be interrogated in their spatial context within the tumor microenvironment (TME). Analysis methods for the resultant complex data are not well formalized, and bespoke methods are usually required to fully capitalize on the underlying richness of information made available by IMC. Deep learning [DL] approaches, while highly accurate for other imaging modalities, have been slow to be adopted in IMC, as public resources for deep learning tasks in IMC are not abundant. Methods: We developed multiple DL and ML-based analysis pipelines for the following tasks in IMC data processing: [1] nucleus and necrotic tissue segmentation, [2] quantitative nuclear and cellular morphometry, [3] identification of cell type-specific niches. We applied these protocols to images and derived single cell spatial data from the TRACERx IMC cohort (n=81 non-small cell lung cancer patients, 561 images). Results: [1] We created a 120 image, 46,000+ labelled nucleus segmentation dataset for IMC data with representative images from lung adenocarcinoma, squamous cell carcinoma and other tissues. We achieved state-of-the-art performance in nuclear instance segmentation using a custom U-net++ neural network architecture trained using this dataset, which we benchmarked against traditional image processing methods, as well as publicly available deep learning architectures. Subsequently, we exploited transfer learning to retrain this model on a restricted dataset of labelled necrotic domains, which produced predictions in good agreement with independent pathologist assessment. [2] We developed an IMC morphometry pipeline utilizing ML-informed partitions of nuclear and cellular shape descriptors through which we performed cell-type specific morphometric characterization of all mapped cells in the non-small cell lung cancer TME, and which enabled a comparative analysis of the morphometries of each distinct cellular phenotype. [3] We established a high throughput density-based spatial clustering pipeline capable of identifying locally enriched niches of a given cell type of interest, as well as probing the composition and phenotypes of other cells within these niches. Conclusions: These approaches enhanced the quality as well as the breadth of spatial information derivable from TRACERx IMC data. Applying such tools to other clinical and pre-clinical datasets can improve our understanding of the spatial organization of cells both in non-small cell lung cancer and other cancer types. Citation Format: Alastair Magness, Katey Enfield, Mihaela Angelova, Emma Colliver, Emer Daly, Kristiana Grigoriadis, Claudia Lee, Oriol Pich, Philip Hobson, Dina Levi, Takahiro Karasaki, David Moore, Julian Downward, Erik Sahai, Mariam Jamal-Hanjani, Charles Swanton, TRACERx Consortium. Machine learning-enhanced image and spatial analytic pipelines for imaging mass cytometry applied to the TRACERx non-small cell lung cancer study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1926.

Label-Free Imaging to Track Reprogramming of Human Somatic Cells.

The process of reprogramming patient samples to human-induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient, leading to a heterogeneous population of cells. In this study, we track the reprogramming status of patient-derived erythroid progenitor cells (EPCs) at the single-cell level during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. EPCs isolated from human peripheral blood of three donors were used for our proof-of-principle study. We found distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide during reprogramming. Random forest models classified iPSCs with ∼95% accuracy, which enabled the successful isolation of iPSC lines from reprogramming cultures. Reprogramming trajectories resolved at the single-cell level indicated significant reprogramming heterogeneity along different branches of cell states. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique, real-time, and nondestructive method to assess the quality of iPSCs in a biomanufacturing process, which could have downstream impacts in regenerative medicine, cell/gene therapy, and disease modeling.

Impact of the Endocannabinoid System on Bone Formation and Remodeling in p62 KO Mice.

Several studies have shown that the G-protein coupled cannabinoid receptor CB2 and its interaction partner p62 are molecularly involved in bone remodeling processes. Pharmacological activation of the CB2 receptor enhanced bone volume in postmenopausal osteoporosis and arthritis models in rodents, whereas knockout or mutation of the p62 protein in aged mice led to Paget’s disease of bone-like conditions. Studies of pharmacological CB2 agonist effects on bone metabolism in p62 KO mice have not been performed to date. Here, we assessed the effect of the CB2-specific agonist JWH133 after a short-term (5 days in 3-month-old mice) or long-term (4 weeks in 6-month-old mice) treatment on structural, dynamic, and cellular bone morphometry obtained by μCT of the femur and histomorphometry of the vertebral bodies in p62 KO mice and their WT littermates in vivo. A genotype-independent stimulatory effect of CB2 on bone formation, trabecular number, and trabecular thickness after short-term treatment and on tissue mineral density after long-term treatment was detected, indicating a weak osteoanabolic function of this CB2 agonist. Moreover, after short-term systemic CB2 receptor activation, we found significant differences at the cellular level in the number of osteoblasts and osteoclasts only in p62 KO mice, together with a weak increase in trabecular number and a decrease in trabecular separation. Long-term treatment showed an opposite JWH133 effect on osteoclasts in WT versus p62 KO animals and decreased cortical thickness only in treated p62 KO mice. Our results provide new insights into CB2 receptor signaling in vivo and suggest that CB2 agonist activity may be regulated by the presence of its macromolecular binding partner p62.

Improved 3D cellular morphometry of Caenorhabditis elegans embryos using a refractive index matching medium.

Cell shape change is one of the driving forces of animal morphogenesis, and the model organism Caenorhabditis elegans has played a significant role in analyzing the underlying mechanisms involved. The analysis of cell shape change requires quantification of cellular shape descriptors, a method known as cellular morphometry. However, standard C. elegans live imaging methods limit the capability of cellular morphometry in 3D, as spherical aberrations generated by samples and the surrounding medium misalign optical paths. Here, we report a 3D live imaging method for C. elegans embryos that minimized spherical aberrations caused by refractive index (RI) mismatch. We determined the composition of a refractive index matching medium (RIMM) for C. elegans live imaging. The 3D live imaging with the RIMM resulted in a higher signal intensity in the deeper cell layers. We also found that the obtained images improved the 3D cell segmentation quality. Furthermore, our 3D cellular morphometry and 2D cell shape simulation indicated that the germ cell precursor P4 had exceptionally high cortical tension. Our results demonstrate that the RIMM is a cost-effective solution to improve the 3D cellular morphometry of C. elegans. The application of this method should facilitate understanding of C. elegans morphogenesis from the perspective of cell shape changes.

Impairment of lymphatic endothelial barrier function by X-ray irradiation

Purpose: Although the microvascular system is a significant target for radiation-induced effects, the lymphatic response to radiation has not been extensively investigated. This is one of the first investigations characterizing the lymphatic endothelial response to ionizing radiation.Materials and methods: Rat mesenteric lymphatic endothelial cells (RMLECs) were exposed to X-ray doses of 0, 0.5, 1, 1.5, and 2 Gy. RMLEC cellular response was assessed 24 and 72-h post-irradiation via measures of cellular morphometry and junctional adhesion markers. RMLEC functional response was characterized through permeability experiments.Results: Cell morphometry showed radiation sensitivity at all doses. Notably, there was a loss of cell-to-cell adhesion with irradiated cells increasing in size and cellular roundness. This was coupled with decreased β-catenin and VE-cadherin intensity and altered F-actin anisotropy, leading to a loss of intercellular contact. RMLEC monolayers demonstrated increased permeability at all doses 24 h post-irradiation and at 2-Gy 72 h post-irradiation.Conclusions: In summary, lymphatics show radiation sensitivity in the context of these cell culture experiments. Our results may have functional implications of lymphatics in tissue, with endothelial barrier dysfunction due to loss of cell-cell adhesion leading to leaky vessels and lymphedema. These preliminary experiments will build the framework for future investigations towards lymphatic radiation exposure response.

Risk Stratification of Oral Potentially Malignant Disorders in Fanconi Anemia Patients Using Autofluorescence Imaging and Cytology-On-A Chip Assay

Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a “cytology-on-a-chip” (COC)–based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients’ OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients’ OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.

<I>Stigonema tagorum</I> Sp. Nov. (Stigonemataceae, Cyanoprokaryota) from a Stone Monument of Santiniketan, West Bengal, India

Cyanobacteria/Cyanoprokaryotes are primary components of the microbial bio-films colonizing on different culturally important stone monuments. Their diversity and eco-physiology was always emphasized as they cause weathering of their substratum leading to disfigurement of the monuments. In the present communication, a novel Stigonematacean species, i.e., Stigonema tagorum is described from a stone monument (Gandhi Statue) in Santiniketan, West Bengal. The taxon has a distinguished type of true branching and cellular morphometry which differentiates it from its resembling taxa.

Micro- and nanotopography with extracellular matrix coating modulate human corneal endothelial cell behavior

The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na+/K+-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na+/K+-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical cues.

Quantitative exfoliative cytology of squames obtained from iron defi ciency anemia and healthy individuals

Background: Iron deficiency anemia is the most common nutritional deficiency of world affecting people of all ages and economic groups. Previous studies on the effects of iron deficiency anemia by exfoliative oral cytology have produced conflicting results but application of quantitative parameters such as nuclear diameter (ND) and cytoplasmic diameter (CD) and nuclear to cytoplasmic ratio (N/C) has shown to be significant in the diagnosis of oral lesions. The present study was undertaken to assess the morphometric changes in CD and ND of squames obtained from buccal mucosa of patients with iron deficiency anemia and also to assess the effect of serum ferritin levels on cellular morphometry. Materials and Methods: Thirty cases of iron deficiency anemia and 30 cases of control group were selected for the study. The control group consisted of people without clinical symptoms of anemia, normal hematological and serum ferritin levels. Scrapings were taken from the buccal mucosa of the iron deficiency anemia patients and control groups and stained with Papanicolaou stain. ND and CD were measured using image analyzer. Results: CD and ND values of the control group were found to be in the range of 65.32-75.39 μ and 8.10-9.40 μ respectively. CD values of iron deficiency were 55.05-64.12 μ with the mean CD value of 59.77 μ and ND values were 8.69-11.24 μ with mean ND values of 9.88 μ. On correlating the serum ferritin and red cell parameters with the CD and ND values of iron deficiency anemia showed positive correlation. Conclusions: The decrease in cytoplasmic diameter and increase in ND in iron deficiency anemia and progressive decrease in CD with decrease in serum ferritin levels suggested that iron deficiency causes significant changes in oral exfoliative cells. Cytomorphometric analysis of smears is useful in detecting the changes in iron deficiency anemia.

Cellular morphometry in late-life depression: a review of postmortem studies.

The impact of major depression in late life is considerable and set to intensify with a worldwide shift in demographic profile toward an elderly population. Although the precise neurobiological mechanisms are not fully understood, a significant body of clinical, epidemiological, and imaging data have suggested divergent pathophysiological pathways underlie depression in late life, when compared with younger patients. Neuroimaging studies have demonstrated significant increases in white matter hyperintensities in late-life depression in several key areas involved in affective circuitry. Postmortem cellular morphometry studies have played a vital role in the identification of discrete changes in the brain microstructure in depression. This review draws together such postmortem studies, which have utilized tissue from younger/mixed age and late-life depressed patients. These findings have suggested varying neuronal and glial cell pathology in depression between different age cohorts. This age-related disparity may suggest different pathophysiological basis for depression, with vascular factors playing a potentially greater role in late life.

A quantitative framework for the 3D characterization of the osteocyte lacunar system

Assessing the role of osteocyte lacunae and the ways in which they communicate with one another is important for determining the function and viability of bone tissue. Osteocytes are able to play a significant role in bone development and remodeling because they can receive nourishment from, interact with, and communicate with other cells. In this sense the immediate environment of an osteocyte is crucial for understanding its function. Modern imaging techniques, ranging from synchrotron radiation-based computed tomography (SR CT) to confocal laser scanning microscopy, produce large volumes of high-quality imaging data of bone tissue on the micrometer scale in rapidly shortening times. These images often contain tens of thousands of osteocytes and their lacunae, void spaces which enclose the osteocytes. While theoretically possible, quantitative analysis of the osteocyte lacunar system is too time consuming to be practical without highly automated tools. Moreover, quantitative morphometry of the osteocyte lacunar system necessitates clearly defined, robust, and three-dimensional (3D) measures. Here, we introduce a framework for the quantitative characterization of millions of osteocyte lacunae and their spatial relationships in 3D. The metrics complement and expand previous works looking at shape and number density while providing novel measures for quantifying spatial distribution and alignment. We developed model, in silico systems to visualize and validate the metrics and provide a concrete example of the attribute being classified with each metric. We then illustrate the applicability to biological samples in a first study comparing two strains of mice and the effect of growth hormone. We found significant differences in shape and distribution between strains for alignment. The proposed quantitative framework can be used in future studies examining differences and treatment effects in bone microstructure at the cell scale. Furthermore, the proposed strategy for quantitative bone cell morphometry will allow investigating structure–function relationships in bone tissue, for example by linking cellular morphometry to bone remodeling.

Three-Dimensional Cellular Morphometry: A New Horizon for Cytology and Cancer Detection

Three-Dimensional Cellular Morphometry: A New Horizon for Cytology and Cancer Detection

Effect of age on the myosin-V immunoreactive myenteric neurons of rats ileum

Alterations in the gastrointestinal neuromuscular function related to age have been demonstrated in human and animal models. This study analyzes the effects of the aging process on the area of the neuronal cell bodies of the myenteric plexus in the antimesenteric and intermediate regions of the ileal circumference of Wistar, 12 month-old in comparison 3 month-old animals. The ileum was removed and whole-mount preparations immunostained by the antibody anti-myosin-V were processed. The morphometric analyses were performed using a computerized image analysis system, with a subsequent distribution of neurons by size in intervals of 100 micro2. The cellular body morphometry revealed a significant increase in the size of the myosin-V- immunoreactive myenteric neurons from 12 month-old animals when compared with 3 month-old animals. However, significant differences between the regions were not observed; these observations were not age-dependent. The implications of these results in relation to the increase of the body weight, size of the small intestine, general organization of the myenteric plexus, staining method of neurons and the possible factors involved in the regulation and/or control of the volume of neronal cells due to aging, are discussed.

Quantitative cytochemistry of cytochrome oxidase and cellular morphometry of the human inferior colliculus in control and Alzheimer's patients

Quantitative cytochemistry of cytochrome oxidase (C.O.) was implemented in human brains to measure C.O. activity in the 3 main divisions of the inferior colliculus (IC): central (ICC), dorsal (ICD), and external (ICE). Units of C.O. activity (μmol/min/g tissue wet weight) were quantified in cellular compartments (overall average, neuropil, perikaryon, and dendrites) at the light microscope level using microdensitometry calibrated with C.O. activity standards measured spectrophotometrically. In a non-AD (Alzheimer's disease) control group (mean age = 79.6 ± 3.1 years, postmortem time = 6.9 ± 1.6 h), the ICC and ICD demonstrated higher ( p < 0.008) overall average activities (mean = 183.40 ± 18.7 and 184.98 ± 45.1 units, respectively) relative to the ICE (56.46 ± 15.9 units). Comparison of cellular morphometry (soma and nucleus area, perimeter, and diameter) revealed that the ICC contained cells of significantly larger soma size than in both the ICD and ICE ( p < 0.002). The distribution of soma diameters in the ICC of controls showed a clear bimodality, enabling the typing of the cells into larger and smaller than average soma diameter. Brains from patients with Alzheimer's disease (AD: mean age = 78.3 ± 2.9 years, postmortem time = 6.5 ± 1.3 h) were compared with the non-AD controls. Significant group differences were found only in the large cells of the ICC. The AD large cells showed a decrement in C.O. activity relative to the corresponding controls in overall average activity ( p < 0.032) and in peak activity of neuropil near the soma ( p < 0.012). These findings provide the first quantitative cytochemical data of C.O. activity in humans. They also suggest that cellular alterations in C.O. metabolism in AD affect predominantly specific groups of larger projection neurons while neighboring neurons are spared.

Cytonuclear morphometry in assesment of dysplasia in polyps Pilot study

In order to objectify the degree of dysplasia, results of nuclear and cellular morphometry were compared with assessed grades of dysplasia in 32 colorectal adenomatous polyps. Of these adenomas 8 showed mild, 17 moderate and 7 severe dysplasia (according to blind duplicate assessments by two pathologists). Using a microscope video-overlay interactive digitizing measurement system, 100 nuclei were measured in each specimen according to a strict measurement protocol. In a stepwise discriminant analysis the best discriminating features appeared to be mean stratification index, N/C ratio, mean contour ratio and mean form AR. Overall with these features 65.6% correct jackknifed classification of the 32 cases could be achieved. On the other hand, a clear three-group distinction could not be obtained, even with the best set of discriminating features. Similarly to gastric dysplasia, the morphometric features might be used to design a two-grade (low, high) rather than a three-grade system to assess the degree of dysplasia. These findings make clear that objectifying the degree of dysplasia in adenomatous polyps is possible by means of interactive morphometric analysis.

Distribution and organization of cholinergic neurons in the rat forebrain demonstrated by computer-aided data acquisition and three-dimensional reconstruction.

An understanding of the organization of cholinergic neurons in the central nervous system has been an important objective for many years. By developing and applying a new electronic method for mapping tissue sections, we have generated original graphic and quantitative findings on forebrain cholinergic neurons that provide new insight into their distribution and organization. Satoh, Armstrong, and Fibiger (Brain Res. Bull. 11:693-720, 1983) have proposed that in the basal forebrain cholinergic neurons with long axons form a continuum rather than being arranged as a series of discrete nuclear groups. It has been difficult, however, by conventional methods of data analysis and display, to test this hypothesis. By using a digital microscopy system, the position of every cholinergic neuron was marked with 1-micron resolution in tissue sections taken at 90-microns or 180-microns intervals through the entire distribution of these neurons in the forebrain. The three-dimensional reconstruction of these neurons in context shows them to be distributed as a continuous cell column. The column twists and changes position as it is deformed by adjacent neuronal structures, such that its shape and continuity would not be apparent without reconstruction into a computer graphics model. Complementary analyses of the distribution of cholinergic interneurons in dopamine-rich regions of the forebrain indicated that there are regional differences between striatal and olfactory tubercle neurons. Cellular morphometry analyses show the population of cholinergic neurons in the rat to be surprisingly homogenous in size, but not in shape. Graphic and quantitative analyses indicated that there is a striking relationship between the distributions of projection and interneuronal cell groups. We conclude that the basal forebrain cholinergic neurons form a continuum. The chemoarchitecture of this cell group does not conform to the usual cytoarchitectural divisions. The present results, however, taken together with the findings based on Nissl-stained sections and connectional and biochemical data, suggest that the region of this neurochemically defined continuum should be reexamined for consideration as a single functional entity or nucleus: a cholinergic basal nuclear complex.

Pulmonary toxicity associated with fly ash from fluidized bed coal combustion: II. Cellular morphometry of distal lung following a single intratracheal instillation

Fly ash from an experimental fluidized bed coal combustor (FBC) was intratracheally instilled (once) into male Syrian golden hamsters at concentrations of 0 (control), 3, 10, 30, and 100 mg/animal. Thirty days postadministration, lungs were fixed by intratracheal perfusion, and tissues were processed for electron microscopic evaluation. Standard point count morphometry was used to analyze distal lung structures. Significant differences were found in volume density of noncellular interstitial space, with this compartment being increased in a dose-dependent manner. Volume density, numerical density, and average cellular volume of distal lung cells revealed no significant differences between treated and control animals. In addition, diffusion capacity and air-blood barrier thickness were not significantly altered in treated animals. The absence of cellular change at 30 days postexposure suggests a residual effect on noncellular interstitium, which may implicate fibrosis.