Imaging of dense cell cultures by multiwavelength lens-free video microscopy.

They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm2 ) over a large field of view of 29.4 mm2 . Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry.

In vivo assessment of tumoral angiogenesis.

Vessel size imaging (VSI) for brain tumor characterization was evaluated and the vessel size index measured by MRI (VSIMRI) was correlated with VSI obtained by histology (VSIhisto). Blood volume (BV) and VSI maps were obtained on 12 rats by simultaneous measurements of R2* and R2, before and after the injection of a macromolecular contrast agent, AMI-227. Immunostaining of collagen IV in vessels was performed. An expression was derived for evaluating VSI from stereologic measurements on histology data (VSIhisto). On BV and VSI images obtained from large-size tumors (n = 9), three regions could be distinguished and correlated well with histological sections: a high BV region surrounding the tumor, a necrotic area where BV is very low, and a viable tumor tissue region showing lower BV but higher VSI than the normal rat cortex, with the presence of larger vessels. The quantitative analysis showed a good correlation (Spearman rank's rho = 0.74) between VSIhisto and VSIMRI with a linear regression coefficient of 1.17. The good correlation coefficient supports VSI imaging as a quantitative method for tumor vasculature characterization.

Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and 'ghost' fibers (all with a diameter of 10-20 micro m) at different (0-3 micro moll(-1)) free Ca(2+) concentrations in the medium. In all these preparations, the apparent K(m) of mitochondrial respiration for exogenous ADP at free Ca(2+) concentrations of 0-0.1 micro moll(-1) was very high, in the range of 250-350 micro moll(-1), in contrast to isolated mitochondria in vitro (apparent K(m) for ADP is approximately 20 micro moll(-1)). An increase in the free Ca(2+) concentration (up to 3 micro moll(-1), which is within physiological range), resulted in a very significant decrease of the apparent K(m) value to 20-30 micro moll(-1), a decrease of V(max) of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent K(m) for ADP (300-350 micro moll(-1)) nor V(max) of respiration changed in the range of free Ca(2+) concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures.

[Magnetic resonance cerebral blood volume maps. Comparison with histologic findings in different types of brain lesions]. / Volume sanguin cérébral déterminé par IRM de perfusion.

Recent developments in magnetic resonance (MR) have made it possible to obtain measurements of the microvasculature within brain lesions. Cerebral blood volume (CBV) maps calculated from dynamic contrast-enhanced MR imaging are particularly sensitive for depicting the microvasculature, and can enable the detection of neovascularization as well as its quantification in relative terms. The purpose of the present work is to compare the results of CBV maps calculated from MR imaging with those from histologic examination of the same region of interest: the biopsy site. Nineteen patients with brain lesions were studied (18 brain tumors and one case of multiple sclerosis). All patients underwent stereotactic biopsy, and calculation of CBV was performed from perfusion MR imaging. Three histopathologic parameters were assessed: the number of vessels (vessel density), the vessel size and the surface area filled by vessels (%). We observed a statistically significant correlation between the vessel density and the CBV, which is consistent with some previous publications. A noninvasive imaging method for characterizing the functional properties, especially hemodynamic activity, of malignant processes seems to be of great benefit to clinical practice.

Identification of membrane calcium channels essential for cytoplasmic and nuclear calcium elevations induced by vascular endothelial growth factor in human endothelial cells.

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the first stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages.

The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system-dependent but ExoU-independent oncosis of neutrophils and macrophages. Time-lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non-chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non-cytotoxic mutant, lacking the type III-secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore-forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact-dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA-infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo-Pro-1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA-induced macrophage death results from a pore-forming activity that is dependent on the intact pcrGVHpopBD operon.

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

BACKGROUND: The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS: After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS: Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS: The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases.

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.

Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells.

The extracellular matrix protein "elastin" is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium- and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton.

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

Effects of intermittent or continuous gravitational stresses on cell-matrix adhesion: quantitative analysis of focal contacts in osteoblastic ROS 17/2.8 cells.

The relationship between cell morphology and cell metabolism and the role of mechanical load in bone remodeling is well known. Mechanical stimulation induces changes in the shape of osteoblasts, probably mediated by reorganization of focal contacts. We studied the influence of gravity (Gz) variations occurring during parabolic flight on osteoblast focal adhesion of ROS 17/2.8 osteosarcoma cells subjected to 15 or 30 parabolic flights. Significant flight-induced shape changes consisted of decreased cell area associated with focal contact plaque reorganization. Identical durations of continuous mechanical stress induced by centrifugation (2 Gz) or clinorotation (Gz randomization) had no major effect on cell focal adhesion. ROS 17/2.8 G2/M synchronization by treatment with nocodazole inhibited the flight-induced decrease in adhesion parameters. We concluded that ROS 17/2.8 cells are sensitive to Gz switches and that their adaptation is at least dependent on microtubule function.

Quantitation of cell-matrix adhesion using confocal image analysis of focal contact associated proteins and interference reflection microscopy.

We have developed an approach for the quantitation of vinculin, a focal contact associated protein, based on a multimodal confocal microscopy and image analysis. Vinculin spot distribution was imaged in confocal fluorescence microscopy and the corresponding focal contacts were imaged in confocal interference reflection microscopy. These images were analyzed with a SAMBA image cytometer. The image analysis program provided 12 morphometric features describing cellular area, shape, and proportions of vinculin spots as well as six topographical features describing the distribution of vinculin and the relative overlap of vinculin and focal contacts. This approach was applied to the study of rat osteosarcoma cells submitted to mechanical stresses: successions of 2g and 0g accelerations during a series of parabolic flights. The measured features were assessed by means of correlation analysis and stepwise discriminant analysis. After correlation analysis, only ten parameters were retained. Quantitation of cell morphological parameters indicated that cell area was significantly affected by gravitational stresses as well as vinculin distribution. Cell area was reduced by 50% and vinculin spots were restricted to cell periphery. Cell adhesion measured by IRM decreased significantly in the first part of the flight and remained stable at the end of the flight. These results suggest that cell-matrix adhesion is affected by gravitational stresses. Image analysis provides useful tools to investigate focal adhesion re-organization under different physiological stimuli.

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to alpha(v)beta3 integrin localization.

This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the alpha(v)beta3 integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the alpha(v)beta3 integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the alpha(v)beta3 integrin distribution.

Quantification of focal contacts in osteoblastic cells--effects of intermittent and continuous gravitational stresses.

NASA: Osteoblast morphology and attachment were studied during parabolic flight and during centrifugation. Cultures of osteosarcoma cells were exposed to gravitational changes and then analyzed for morphological changes and stained using immunofluorescence staining for vinculin. Changes in cell adhesion parameters and focal contact topography are presented and discussed.^ieng

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Mapping of the orientation of myocardial cells by means of polarized light and confocal scanning laser microscopy.

The study of the topological organisation of myocardial cells is a basic requirement for the understanding of the mechanical design of the normal and pathological heart. We developed a technique based on multiparametric image analysis of transmitted polarized light to generate maps of the azimuth and the elevation angles of the myocardial cells. The properties of birefringence of the myocardium embedded in methylmetacrylate were measured in papillary muscles with monitored 3D orientation. This birefringence is positive uniaxial with a 0 degree extinction angle when the axis of the fiber is parallel to the axis of the polarizer or the analyzer. Thick sections were studied between crossed polars, and four images of each section were digitized for an angle of the polarizer with the section varying from 0-67.5 degrees in steps of 22.5 degrees. The amounts of transmitted light for each setup of the polarizer were combined in order to extract the values of the azimuth angle (modulo 90 degrees) and the elevation angle of the myocardial cells, according to the Johannsen equation. The respective maps of these angles were calculated and then assessed with confocal scanning laser microscopy. This method provides an efficient and accurate tool for the study of the histological architecture of the fetal and neonatal heart.

Improvements in visualisation and localisation of human papillomavirus DNA in CaSki cells by fluorescence in situ hybridization, laser scanning confocal microscopy and three-dimensional image reconstruction.

The visual interpretation and localisation of specific DNA sequences in three dimensions in cell nuclei was investigated by fluorescence in situ hybridization (FISH) and laser scanning confocal microscopy (LSCM) using CaSki cells containing 600 copies per cell of human papillomavirus (HPV) DNA type 16 integrated in cellular DNA. Biotinylated DNA probes were used and DNA-DNA hybrids were revealed by a three-step reaction involving a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. The DNA from cell nuclei was counterstained with propidium iodide. With standard fluorescence microscopy, some dense fluorescent spots were seen in the cell nuclei. Similarly, with LSCM, some hybridization spots were observed in the cell nuclei but they were at different levels of the nuclei as shown by successive nuclear sections taken along the z axis. The visualisation of multiple hybridization spots confirmed the presence of multiple integration sites of HPV 16 DNA in CaSki cells. Association of LSCM with three-dimensional reconstructions lead to spatial images of hybridization spots obtained by stacking (x,y) images from consecutive confocal planes. Rotation of the reconstructed cell nuclei around the y axis makes it possible to distinguish closely adjacent spots. The combination of these techniques improves the detection of hybridization spots and may be of interest to further determine whether the HPV DNA is episomal or integrated in infected cells.

Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.

Morphometry of human nerve biopsies by means of automated cytometry: assessment with reference to ultrastructural analysis.

The morphometric analysis of myelinated fibres is a tool for diagnosis of neuropathies and for assessing nerve regeneration after reconstructive surgery. In order to obtain reliable information, sampling techniques should be avoided and the measurement of all the fibres within a nerve fascicle requiring the use of automated cytometry is necessary. We have developed a programme on the SAMBA cytological image analyser for the automated measurement of myelinated fibres. Different techniques of segmentation were tested and adaptive grey level thresholding gave the more reliable result. Accuracy and reproducibility were tested using a set of five human superficial peroneal nerve biopsies that were previously analysed by means of semi-automatic ultrastructural morphometry. A maximum difference of 9% in the number of fibres counted was obtained when comparing the automatic and the semi-automatic methods. In all cases the histograms of the morphometrical variables (fibre and axon diameters, myelin sheath thickness) were found to be identical to the reference histograms obtained by the semi-automatic method.

An automatic method for bone histomorphometry: assessment with reference to usual static and dynamic parameters.

To perform a fast and reproducible analysis in bone histomorphometry, we developed an automatic method for calculating static and dynamic parameters. A color automatic image analyzer (SAMBA 200) was used to obtain the usual parameters of bone histomorphometry: bone volume (Cn-BV%TV), osteoid volume (Cn-OV%BV), and osteoid surface (Cn-OS%BS). A specialized algorithm was designed for calculation of the mineral apposition rate (MAR). Eroded surface (Cn-ES%BS) was read in a semiautomatic mode using a cursor. To validate this program, we input 30 samples from patients with bone disease (20 osteoporosis, 6 renal osteodystrophy, 2 osteomalacia, and 2 hyperparathyroidism) using manual and automatic modes. The results obtained showed a highly significant correlation with the usual manual method for all parameters: OS/BS, r = 0.93; OV/BV, r = 0.98; MAR, r = 0.90. With the automatic method, larger values were found for osteoid parameters and MAR and lower values for BV/TV. There were no statistical differences for OV/BV and MAR when compared to the reference manual method. This study establishes that automatic measurements of osteoid parameters and MAR can be performed by a fast analyzer with as good reproducibility and accuracy as the manual method.