Fluorophore spectroscopy in aqueous glycerol solution: the interactions of glycerol with the fluorophore.

A common perception exists that glycerol provides an inert-like environment modifying viscosity and index of refraction by its various concentrations in aqueous solution. Said perception is herein challenged by investigating the effects of the glycerol environment on the spectroscopic properties of fluorescein, as a representative fluorophore, using steady-state and time-resolved techniques and computational chemistry. Results strongly suggest that the fluorescence quantum yield, measured fluorescence lifetime (FLT), natural lifetime and calculated fluorescence lifetime are all highly sensitive to the presence of glycerol. Glycerol was found to impact both the ground and first excited states of fluorescein, quenching and modifying both absorption and emission spectra, affecting the fundamental electrical dipoles of the ground and first excited singlet states, and lowering FLT and quantum yield. Furthermore, the Stern-Volmer, Lippert-Mataga, Perrin and Strickler-Berg relations indicate that glycerol acts upon fluorescein in aqueous solution as a quencher and alters the fluorescein geometry. Predictions made by computational chemistry impressively correspond to experimental results, both indicating changes in the properties of fluorescein at around 35% v/v aqueous glycerol, a clear indication that glycerol is not an innocent medium. This study proposes the Strickler-Berg relation as a means of detecting non-negligible effects of a hosting medium on its host fluorophore. These new insights on the molecular structures, the interactions between glycerol and its host fluorophore, and the effects of one on the other may be essential for understanding fundamental phenomena in chemistry and related fields.

Discrimination of leukemic Jurkat cells from normal lymphocytes via novo label-free cytometry based on fluctuation of image gray values.

We introduce a simple, label-free cytometry technique, based on the spatio-temporal fluctuation analysis of pixel gray levels of a cell image utilizing the Gray Level Information Entropy (GLIE) function. In this study, the difference in GLIE random fluctuations and its biophysical etiology in a comparison cell model of leukemic Jurkat cells and human healthy donor lymphocytes was explored. A combination of common bright field microscopy and a unique imaging dish wherein cells are individually held untethered in a picoliter volume matrix of optical chambers was used. Random GLIE fluctuations were found to be greater in malignant Jurkat cells than in benign lymphocytes, while these fluctuations correlate with intracellular vesicle Mean Square Displacement (MSD) values and are inhibited by myosin-2 and adenosine triphosphate (ATP) inhibitors. These results suggest that the incoherent active forces acting on the cytoskeleton which cause mechanical dissipative fluctuation of the cytoskeletal and related intracellular content are the biophysical cellular mechanism behind the GLIE random fluctuation results. Analysis of the results in Jurkat cells and normal lymphocytes suggests the possible potential of this simple and automated label-free cytometry to identify malignancy, particularly in a diagnostic setup of multiple cell examination.

The cationic dye basic orange 21 (BO21) as a potential fluorescent sensor.

The present study investigates the fluorescence properties of BO21 and their dependence on various intracellular conditions. The results obtained with cell-free solutions indicate that the influences of pH and temperature on the fluorescence spectra are negligible, while viscosity, various proteins and heparin have significant influence. In the presence of heparin, a red shift of the emission spectrum (from 515 to 550 nm) is observed, suggesting that this shift cannot simply be attributed to electrostatic interaction between BO21 and the polyanionic heparin, but rather to aggregation of BO21 on the polyanion. In water, the quantum yield of BO21 was found to be 1000 times lower than that of fluorescein, yet surprisingly its fluorescence polarization (FP) was found to be about 40 times higher (FP = 0.470), even though both have similar structures and molecular weights. A thorough analytical and experimental investigation of these phenomena indicates that the very high FP of BO21 in water is a consequence of its very short lifetime. However, upon the addition of heparin to aqueous BO21, the fluorescence lifetime (FLT) of BO21 increases from τ = 10.35 to 56.5 ps, with a consequent dramatic drop in its fluorescence polarization from 0.470 to 0.230. From its behavior in aqueous glycerol solution, it is hypothesized, with support from theoretical calculations, that BO21 is a molecular rotor. Using these properties, BO21 may be a good candidate as a sensor, for example, of heparin levels in blood or of intracellular viscosity.

Analysis of the Spectroscopic Aspects of Cationic Dye Basic Orange 21.

Spectroscopic properties of cationic dye basic orange 21 (BO21) in solutions, in solids, and within leukocytes were examined. Results obtained with solutions indicate that influence of variables such as pH, viscosity, salt composition, and various proteins on the absorption spectrum of BO21 is negligible. However, in the presence of heparin, a blue shift (484-465 nm) is observed, which is attributed to the aggregation of BO21 on the polyanion. Applying density functional theory demonstrates that in aqueous solutions (a) the formation of BO21 oligomers is thermodynamically favorable, they are oriented in an antiparallel dipolar arrangement, and their binding energies are lower than those of parallel dipolar arrangements, (b) association between BO21 aggregates and heparin is highly favorable, and (c) the blue shift is due to the mixing of π → π* transitions caused by BO21 molecule stacking. However, when embedded in basophils, the absorption spectra of intracellular BO21 is extremely red-shifted, with two peaks (at 505 and 550 nm) found to be attributed to BO21 and the BO21-heparin complex, respectively, which are intracellularly hosted in nonaqueous environments. Initial evidence of the ability to differentiate between leukocyte types by BO21 is presented.

Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array.

Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities-pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion.

Measurement of Water Saturation in Soybean Oil.

In a previous study, it was observed that survivability was low when attempting to cryopreserve sperm cells in a nanoliter-sized droplet protected under soybean oil, in stark contrast to the high survival rates in milliliter-sized droplets. In this study, infrared spectroscopy was used to provide an estimate of the saturation concentration of water in soybean oil. By following the time evolution of the infrared absorption spectrum of water-oil mixtures, the saturation of water in soybean oil was found to reach equilibrium after 1 h. From the absorption spectra of neat water and neat soybean oil and the application of the Beer-Lambert law to an estimation of the absorption of a mixture from its individual components, it was estimated that the saturation concentration of water is 0.010 M. This estimate was supported by molecular modeling using the latest semiempirical methods (in particular, GFN2-xTB). While for most applications the very low solubility has little impact, the implications in those exceptions were discussed.

Spatial-temporal correlations in the speckle pattern for the characterization of cellular motion within a 3D object.

Dynamic light scattering analysis has been demonstrated recently to be a promising tool for the assessment of structural changes taking place inside opaque tissue samples. Specifically, quantification of velocity and direction of cellular motion inside spheroids and organoids has attracted much attention as a potent indicator in personalized therapy research. Here, we propose a method for the quantitative extraction of cellular motion, velocity, and direction, by applying a concept of speckle spatial-temporal correlation dynamics. Numerical simulations and experimental results obtained on phantom and biological spheroids are presented.

Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device.

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

Co-culture hydrogel micro-chamber array-based plate for anti-tumor drug development at single-element resolution.

In response to the need for reliable cellular models that reflect complex tumor microenvironmental properties, and enable more precise testing of anti-cancer therapeutics effects on humans, a co-culture platform for in-vitro model that enhances the physiology of breast cancer (BC) microenvironment is presented. A six well imaging plate wherein each macro-well contains several separate compartments was designed. Three-dimensional (3D) cancer spheroids are generated and cultured in the inner compartment which is embossed with an array of nano-liter micro-chambers made of hydrogel. Stromal cells are cultured in the outer chambers. The two cell types are cultured side-by-side, sharing a common space, thus enabling extra-cellular communication via secreted molecules. As proof of concept, a model of BC tumor microenvironment was recapitulated by co-cultivating 3D MCF7 spheroids in the presence of tumor-associated macrophages (TAMs). The presence of TAMs induced an aggressive phenotype by promoting spheroid growth, enhancing survivin expression levels and enabling invasive behavior. Moreover, TAMs influenced the response of BC spheroids to cytotoxic treatment as well as hormonal drug therapy, and enhanced the effects of nitric oxide donor. The platform enables time-lapse imaging and treatment without losing spatial location of the measured spheroids, thereby allowing measurements and analysis at individual-object resolution in an easy and efficient manner.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: methodology.

BACKGROUND: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. RESULTS: The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing--thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing--thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. CONCLUSIONS: The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing--thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: functional activity of cryopreserved cells.

BACKGROUND: The cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells. RESULTS: In the present study, U937 cells underwent the freezing and thawing cycle in the i3C device. Then a panel of vital tests was performed, including the number of dead cells (PI staining), apoptotic rate (Annexin V staining), mitochondrial membrane potential (TMRM staining), cytoplasm membrane integrity and intracellular metabolism (FDA staining), as well as post-thawing cell proliferation assays. Cells that underwent the freezing - thawing cycle in i3C devices exhibited the same functional activity as control cells. Moreover, the combination of the multi-parametric analysis at a single cell resolution and the optical and biological features of the device enable an accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells. CONCLUSIONS: The means and methodologies described here enable the freezing and thawing of spatially identifiable cells, as well as the efficient detection of viable, specific, highly biologically active cells for future applications.

Functional analysis of individual cells and microenvironment of breast cancer-draining lymph nodes.

The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.

Intracellular esterase activity in living cells may distinguish between metastatic and tumor-free lymph nodes.

BACKGROUND: One of the major clinical problems in breast cancer detection is the relatively high incidence of occult lymph node metastases undetectable by standard procedures. Since the ascertainment of breast cancer stage determines the following treatment, such a "hypo-diagnosis" leads to inadequate therapy, and hence is detrimental for the outcome and survival of the patients. The purpose of our study was to investigate functional metabolic characteristics of living cells derived from metastatic and tumor-free lymph nodes of breast cancer (BC) patients. METHODS: Our methodology is based on the ability of living cells to hydrolyze fluorescein diacetate (FDA) by intracellular esterases and on the association of FDA hydrolysis rates with a specific cell status, both in physiological and pathological conditions. RESULTS: The present study demonstrates a significant difference in the ability to utilize FDA by lymph node cells derived from metastatic and tumor-free lymph nodes in general average, as well as in the metastatic and tumor-free lymph nodes of individual patients. Cells from metastatic lymph nodes had a higher capacity for FDA hydrolysis, and increased this activity after additional activation by autologous tumor tissue (tt). The association between increased FDA hydrolysis rate and activated T lymphocytes and antigen-presenting cells (APC) was shown. CONCLUSION: The results of the present study may contribute to predicting the risk of involvement of seemingly "tumor-free" axillary lymph nodes in occult metastatic processes, and to reducing false-negative results of axillary examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Lymphocyte resistance to lysophosphatidylcholine mediated apoptosis in atherosclerosis.

OBJECTIVE: Apoptosis is being increasingly regarded as a key component in the development and progression of atherosclerosis. Since it has become apparent that the immune system plays a predominant role in mediating atherogenesis, there has been a growing recognition that the evaluation of lymphocyte apoptosis may contribute to understanding a persistent altered immune and inflammatory response. The aim of the present study was to evaluate the apoptotic effect of lysophosphatidylcholine (LPC) on peripheral blood lymphocytes (PBL) derived from unstable angina (UA) patients, as compared to healthy donors. METHODS: PBL isolated from 27 healthy donors and 25 age matched UA patients were examined. Early apoptotic events induced by LPC in resting and phytohemagglutinin (PHA)-activated lymphocytes were evaluated by several apoptotic assays. The levels of intracellular reactive oxygen species (ROS) and the expression of apoptotic regulated proteins (Bcl-2 and Bax) were measured. RESULTS: LPC was found to induce apoptosis in normal activated lymphocytes, in a dose- and time-dependent manner, in association with an increase in intracellular ROS. In UA patients, an exposure of PHA-activated PBL to LPC triggered neither an increase in ROS generation, nor in the apoptotic manifestations, and was associated with a significantly lower ratio of Bax/Bcl-2 expression. CONCLUSION: Our results indicate that PBL isolated from UA patients may be resistant to apoptosis induction by LPC, resulting from oxidative stress challenge and dysregulation of apoptosis-related protein expression.

Breast cancer detection by Michaelis-Menten constants via linear programming.

The Michaelis-Menten constants (K(m) and V(max)) operated by linear programming, were employed for detection of breast cancer. The rate of enzymatic hydrolysis of fluorescein diacetate (FDA) in living peripheral blood mononuclear cells (PBMC), derived from healthy subjects and breast cancer (BC) patients, was assessed by measuring the fluorescence intensity (FI) in individual cells under incubation with either the mitogen phytohemagglutinin (PHA) or with tumor tissue, as compared to control. The suggested model diagnoses three conditions: (1) the subject is diseased, (2) the diagnosis is uncertain, and (3) the subject is not diseased. Out of 50 subjects tested, 44 were diagnosed correctly, in 5 cases the diagnosis was not certain, and 1 subject was diagnosed incorrectly.

Nitric oxide is cytoprotective to breast cancer spheroids vulnerable to estrogen-induced apoptosis.

Estrogen-induced apoptosis has become a successful treatment for postmenopausal metastatic, estrogen receptor-positive breast cancer. Nitric oxide involvement in the response to this endocrine treatment and its influence upon estrogen receptor-positive breast cancer progression is still unclear. Nitric oxide impact on the MCF7 breast cancer line, before and after estrogen-induced apoptosis, was investigated in 3D culture systems using unique live-cell imaging methodologies. Spheroids were established from MCF7 cells vulnerable to estrogen-induced apoptosis, before and after exposure to estrogen. Spheroids derived from estrogen-treated cells exhibited extensive apoptosis levels with downregulation of estrogen receptor expression, low proliferation rate and reduced metabolic activity, unlike spheroids derived from non-treated cells. In addition to basic phenotypic differences, these two cell cluster types are diverse in their reactions to exogenous nitric oxide. A dual effect of nitric oxide was observed in the breast cancer phenotype sensitive to estrogen-induced apoptosis. Nitric oxide, at the nanomolar level, induced cell proliferation, high metabolic activity, downregulation of estrogen receptor and enhanced collective invasion, contributing to a more aggressive phenotype. Following hormone supplementation, breast cancer 3D clusters were rescued from estrogen-induced apoptosis by these low nitric oxide-donor concentrations, since nitric oxide attenuates cell death levels, upregulates survivin expression and increases metabolic activity. Higher nitric oxide concentrations (100nM) inhibited cell growth, metabolism and promoted apoptosis. These results suggest that nitric oxide, in nanomolar concentrations, may inhibit estrogen-induced apoptosis, playing a major role in hormonal therapy. Inhibiting nitric oxide activity may benefit breast cancer patients and ultimately reduce tumor recurrence.

A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells.

The importance of research involving non-adherent cell lines, primary cells and blood cells is generally undisputed. However, the task of investigating the complexity and heterogeneity of these cells calls for their long-run monitoring at a single-cell resolution. Such a capability is currently unavailable without having to use disruptive cell tethering. The present Cell Retainer (CR) concept enables high-content correlative multi-parametric measurements, from the functional to molecular level, of the same living individual non-adherent cells within a population. Thereby, despite extensive long-term bio-manipulations, the cells preserve their identity without tethering. Several exemplary experiments, using a microscope-slide-based version of the CR, are presented, which could not be performed by other state of the art methods.

Concomitant real-time monitoring of intracellular reactive oxygen species and mitochondrial membrane potential in individual living promonocytic cells.

Reactive oxygen species (ROS) have recently been shown to be involved in multiple physiological responses through modulation of signaling pathways. Inappropriate production of these radicals, and their metabolites, leads to the development of various pathologies. Free radicals can induce both positive and negative effects in cells, and their metabolic pathways are very complex. Hence, it is crucial to be able to simultaneously and directly determine their production dynamics and concentrations in individual living cells, in physiological or pathological states, and in response to drugs. The aim of the present study was to monitor in real time the rates of ROS generation in promonocytic cells upon stimulation with hydrogen peroxide and oxidized lipid. Quantitative detection of intracellular ROS concentration in intact living U937 cells was performed by fluorescence intensity (FI) and polarization (FP) measurements utilizing the Optical LiveCell Array technology. The "dihydro" derivative probes of fluorescein (DCF-DA) and rhodamine (DHR123) were used to assess the intracellular levels of ROS. Each probe molecule exhibited a characteristic FI and FP in its non-fluorescent or oxidized form. Analysis of the temporal relationship between the kinetics of ROS generation and the onset of changes in mitochondrial membrane potential shows high variability within a cell population with regard to both processes. The data demonstrated that temporal measurement of ROS generation, in identifiable individual cells, reveals kinetic behavior that otherwise would be undetected.

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.