Objective identification of cell cycle phases and subphases by automated image analysis.

Frequency distributions of integrated optical density, perimeter, projection, area, form factor, average optical density, and mean dispersion path of nuclear images of Feulgen-stained HeLa S3 cells were obtained by automated image analysis at the base threshold of 0.04 OD. The mean values and standard deviations of these geometric parameters were then computed versus increasing values of threshold (0.08--0.32 OD). There is clear evidence of differential chromatin dispersion and convolution during the cycle of synchronized HeLa S3 cells at different times after selective mitotic detachment. The combination of average OD, form factor, and mean dispersion path at base threshold with the threshold dependence of nuclear morphometric parameters permits objective identification of cell cycle phases and their subphases, by characterizing variations in chromatin geometry within and between phases, regardless of whether DNA content remains constant (early G1, middle G1, late G1), varies only slightly (late G1-early S or late S-G2 transitions), or varies significantly (early S-middle S).     

Multiparametric cell cycle analysis by automated microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.     

Cell cycle variations in chromatin structure detected by DNase I

We have recently developed a reproducible method for the use of DNase I as a sensitive probe of chromatin structure (Prentice, D A & Gurley, L R, Biochim biophys acta 740 (1983) 134) [12] and have used this probe to investigate chromatin structure during the interphase of the cell cycle. Chinese hamster cells (line CHO) were synchronized by: (1) mitotic detachment, to obtain M-phase cells; (2) isoleucine deprivation, to obtain G1-phase cells; and (3) sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea, to obtain cells blocked at the start of S phase. The cells were released from the various blocking schemes and nuclei were isolated and digested with DNase I at various times. The digestion kinetics were monitored to detect possible changes in chromatin condensation through the cell cycle. The chromatin was much more accessible to DNase I in G1 phase than in S or G2 phase, with only small variations in structure detected in late G1 and very early S phase. From early S phase up to mitosis, the chromatin became increasingly condensed and inaccessible to DNase I action. These results support the concept of a chromatin condensation cycle during interphase as well as during mitosis.     

Effect of hyperbaric stress on yeast morphology: study by automated image analysis

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO₂.     

Bone cell elasticity and morphology changes during the cell cycle

The mechanical properties of cells are reported to be regulated by a range of factors including interactions with the extracellular environment and other cells, differentiation status, the onset of pathological states, as well as the intracellular factors, for example, the cytoskeleton. The cell cycle is considered to be a well-ordered sequence of biochemical events. A number of processes reported to occur during its progression are inherently mechanical and, as such, require mechanical regulation. In spite of this, few attempts have been made to investigate the putative regulatory role of the cell cycle in mechanobiology. In the present study, Atomic Force Microscopy (AFM) was employed to investigate the elastic modulus of synchronised osteoblasts. The data obtained confirm that osteoblast elasticity is regulated by cell cycle phase; specifically, cells in S phase were found to have a modulus approximately 1.7 times that of G1 phase cells. Confocal microscopy studies revealed that aspects of osteoblast morphology, namely F-actin expression, were also modulated by the cell cycle, and tended to increase with phase progression from G0 onwards. The data obtained in this study are likely to have implications for the fields of tissue- and bio-engineering, where prior knowledge of cell mechanobiology is essential for the effective replacement and repair of tissue. Furthermore, studies focused on biomechanics and the biophysical properties of cells are important in the understanding of the onset and progression of disease states, for example cancer at the cellular level. Our study demonstrates the importance of the combined use of traditional and relatively novel microscopy techniques in understanding mechanical regulation by crucial cellular processes, such as the cell cycle.     

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.     

Analysis of the cell kinetics during planarian regeneration by means of SAMBA 200 cell image processor

Summary The relationships between cell kinetics and nuclear transformations in regeneration were investigated in the planarianPolycelis nigra by means of image analysis. A SAMBA 200 cell image processor was used to compute densitometric, textural and morphological parameters on Feulgen-stained nuclei in the blastema and near the cut 2–96 h after decapitation. On the basis of these parameters, the phase of the cell cycle (G₁–G₀, S, G₂ and M) was identified and the variations in the percentage of cells in the various phases as well as the blastema cell number were computed against time after decapitation. It was demonstrated that the transection is followed by the sequential wasting of the M, G₂, S and G₁–G₀ compartments. The depletion of a compartment was interpreted as being responsible for the subsequent recovery observed in the next one. The results show that cell proliferation at the section level is not sufficient to account for the increase of the blastema cell number during the first 48 h of regeneration, since the doubling time is about 12 h while the average cycle time is 48 h. It is thus suggested that G₁–G₀ cells migrate toward the section level, at least during the first 2 days of regeneration. Analysis of the nuclear profiles demonstrated that there are two different classes of G₁–G₀ cells: one corresponding to mature cells with a lot of condensed chromatin distributed in clumps within the nucleus, the other to immature cells with chromatin regularly distributed according to a rather homogeneous pattern. About one G₁–G₀ cell out of five is immature at the section level before decapitation while four cells out of five are immature as early as 8 h after the cut. This early inversion of the ratio between mature and immature cells argues in favour of an immigration of immature G₁–G₀ cells to the young blastema, where they are expected to accomplish only one cell cycle, and thus gives rise to mature cells.     

Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase

The ataxia telangiectasia-mutated (ATM) and Rad3-related kinase (ATR) is a central component of the cell cycle checkpoint machinery required to induce cell cycle arrest in response to DNA damage. Accumulating evidence suggests a role for ATR in signaling DNA damage during S-phase. Here we show that ATR is recruited to nuclear foci induced by replication fork stalling in a manner that is dependent on the single stranded binding protein replication protein A (RPA). ATR associates with chromatin in asynchronous cell cultures, and we use a variety of approaches to examine the association of ATR with chromatin in the absence of agents that cause genotoxic stress. Under our experimental conditions, ATR exhibits a decreased affinity for chromatin in quiescent cells and cells synchronized at mitosis but an increased affinity for chromatin as cells re-enter the cell cycle. Using centrifugal elutriation to obtain cells enriched at various stages of the cell cycle, we show that ATR associates with chromatin in a cell cycle-dependent manner, specifically during S-phase. Cell cycle association of ATR with chromatin mirrors that of RPA in addition to claspin, a cell cycle checkpoint protein previously shown to be a component of the replication machinery. Furthermore, association of ATR with chromatin occurs in the absence of detectable DNA damage and cell cycle checkpoint activation. These data are consistent with a model whereby ATR is recruited to chromatin during the unperturbed cell cycle and points to a role of ATR in monitoring genome integrity during normal S-phase progression.     

Modulation of cytochemically detected dihydrofolate reductase during cell cycle.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an important enzyme involved in DNA metabolism. In this connection the cell cycle modulation of DHFR levels in HeLa S3 and HL 60 cell lines was investigated by flow cytometric analysis. A concentration of 4 micrograms/ml of aphidicolin was employed to synchronize the cell lines. DHFR was cytochemically detected by using tetrazolium salt and immunofluorescence techniques; DNA content was evaluated by means of propidium iodide staining. At 0, 2, 4, 6, 8, 10, 12 hrs. after the removal of the drug we observed a low DHFR level in G0-G1 phase, followed by an increase during late S and G2/M phases. The variations of this enzyme may represent, under well defined conditions, a marker of cycling cells.     

Assessment of cell proliferation on porous microcarriers by means of image analysis

Spherical porous microcarriers (PMCs) made from collagen-glycosaminoglycan crosslinked copolymers have exhibited considerable promise as growth surfaces for the proliferation of anchorage-dependent mammalian cell lines and have demonstrated the ability to entrap anchorage-independent cells. However, quantification of cell growth on PMCs has proved difficult. A method of measuring the proliferation of PMCs, based on image analysis, is presented. Using CV1 and CHO cell lines, samples of PMCs were removed from culture at various times, fixed, embedded and sectioned. The 2 microns sections were stained, photographed and digitized in three colors. A computer program was developed to evaluate digitized PMC cross-sections and to classify pixels as conforming to either background, cytoplasmic, matrix or nuclear parameters, based on a set of classification rules determined by statistical analysis. Growth curves were generated by relating the number of pixels occupied by cellular material to the total number of pixels in the PMC cross-section. The PMCs were found to foster cell proliferation, with cell densities approaching 100% occupancy.     

TOUSLED kinase activity oscillates during the cell cycle and interacts with chromatin regulators

The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism.     

Topology-based fluorescence image analysis for automated cell identification and segmentation

Cell segmentation refers to the body of techniques used to identify cells in images and extract biologically relevant information from them; however, manual segmentation is laborious and subjective. We present Topological Boundary Line Estimation using Recurrence Of Neighbouring Emissions (TOBLERONE), a topological image analysis tool which identifies persistent homological image features as opposed to the geometric analysis commonly employed. We demonstrate that topological data analysis can provide accurate segmentation of arbitrarily-shaped cells, offering a means for automatic and objective data extraction. One cellular feature of particular interest in biology is the plasma membrane, which has been shown to present varying degrees of lipid packing, or membrane order, depending on the function and morphology of the cell type. With the use of environmentally-sensitive dyes, images derived from confocal microscopy can be used to quantify the degree of membrane order. We demonstrate that TOBLERONE is capable of automating this task.     

Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis

Many filamentous bacteria and fungi tend to form pellets, or mixtures of dispersed mycelium and pellets in liquid fermentation broths. In some cases, a specific kind of morphology is required for optimum product yield. When quantitative analysis and characterization of the pellet morphology are needed, an image processing system can be used. It allows a fast and reproducible analysis of the frequency distribution of pellet size, mean pellet size, contents of pellets, or their shape. The use of such a system allows for an on-line analysis. For a demonstration of the method, results of two fermentations of Streptomyces tendae are shown.     

Coupling of chromatin structure to cell geometry during the cell cycle: Transformed versus reverse-transformed CHO

The cellular and nuclear morphometry of synchronized CHO cells growing on glass in the presence or absence of 10⁻³M of cAMP has been determined. Measurements were made at various time intervals following selective mitotic detachment. The changes in nuclear morphometry during the cycle of cells growing on glass confirms previous findings on similar cells growing in suspension. When cells are grown on glass, the cell shape also changes. Specifically, when cells are treated with cAMP, the cytoplasmic cell shape and condensation is directly coupled with nuclear chromatin shape and condensation, but they are uncoupled for the transformed cells.     

Changes in chromatin compaction during the cell cycle revealed by micrometer-scale measurement of molecular flow in the nucleus

We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca(2+) intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes.     

Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry.

BACKGROUND: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. AIM: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. METHODS: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn ADP Flow Cytometer. RESULTS: The pHi clamping conditions were optimized as 140 mM potassium and 10 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. CONCLUSION: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.     

Changes in chromatin structure during the mitotic cycle

Summary Optical density profiles of Feulgen-stained nuclei ofBryonia dioica at different stages of the mitotic cycle were determined. Nuclei in the G₂ phase have a greater fraction of dense chromatin than nuclei in G₁ phase. However, nuclei at the end of the S phase have dispersed chromatin of minimal density. Thus, chromatin density oscillates during the mitotic cycle of this species, consequently the progressive increase in density previously recorded throughout the intermitotic period of two other species (onion and mouse) cannot be a general rule.