Cellular Dry Mass Research Articles

Label-Free Digital Holographic Microscopy for In Vitro Cytotoxic Effect Quantification of Organic Nanoparticles.

Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.

Photosynthesis, Growth and Dry Biomass Production in Euglena gracilis Klebs as Affected by Three Growth Irradiance Levels

Aims: The research aimed to investigate the shade response of E. gracilis Klebs while making the irradiance a crucial factor for photosynthesis based physiological activities and its applicability for industrial level culture conditions.
 Study Design: Euglena was cultured at three different light intensities of 30, 90, and 210 mol m-2s-1 photoautotrophically and axenically in modified Cramer-Meyer medium at 25 ˚C as batch cultures.
 Methodology: The photosynthesis O2 evaluation of Euglena cultures was measured under exponential (EP), transitional (TP), and stationary phases (SP). The light compensation point (LCP), light saturation point (LSP), and dark respiration rate (DRR) were obtained. Cell volume and cell number in each culture were measured simultaneously. Cells were collected and obtained dry mass (DM) after drying aliquots at 80˚C. Specific growth rate (SGR) and relative growth rate (RGR) were calculated. Tests for homogeneity of variance were performed on all parameters and LSDs were used for the mean separation.
 Results: In the TP, the lowest LCP was achieved in the higher light culture. The values of both the DRR and the LSP were the same as in EP. The DRR, LCP and LSP are lower in lower PFD cultures and decreased with increasing cell titers. The cellular growth levels were lower in lower light culture and decreased as each culture grew. Cellular DM was maintained constant in the EP, where SGR almost equaled RGR. In the EP, SGR was maintained constant in each culture, SGR displayed a saturation phenomenon. In the later TP, SGR became equal to RGR and all the cultures revealed constant DM.
 Conclusion: Euglena photoautotrophic cultures can tolerate low light intensities. With the SGR and RGR behavior under the shade conditions, they can maintain the constant photosynthesis rate and constant dry matter level.

Significant difference in response of malignant tumor cells of individual patients to photodynamic treatment as revealed by digital holographic microscopy.

The investigation of in-vitro response of cell cultures derived from tumor material of individual patients with similar tumor localizations to photodynamic treatment is presented. Tumor types included in the research were renal cell carcinoma, melanoma and alveolar, synovial, lypo- and osteo- sarcomas. Long-term observations of treatment-induced morphological changes in cells were performed by means of digital holographic microscopy. A substantial variance in response of cells of individual patients with similar tumor types and localizations to photodynamic treatment with the same dose has been observed. These peculiarities are indicative of the demand to personalized protocols of photodynamic treatment. The elevated resistance of cells of some patients to treatment at high doses highlights potential limitations of photodynamic therapy for some patients. Digital holographic microscopy is shown to be an informative label-free noninvasive tool allowing for long-term monitoring of cell samples in vitro and providing quantitative information on necrosis rate and loss of cellular dry mass. The developed methodology can be generalized for analysis of cellular response to various therapies.

Multiscale Assay of Unlabeled Neurite Dynamics Using Phase Imaging with Computational Specificity.

Primary neuronal cultures have been widely used to study neuronal morphology, neurophysiology, neurodegenerative processes, and molecular mechanism of synaptic plasticity underlying learning and memory. However, the unique behavioral properties of neurons make them challenging to study, with phenotypic differences expressed as subtle changes in neuronal arborization rather than easy-to-assay features such as cell count. The need to analyze morphology, growth, and intracellular transport has motivated the development of increasingly sophisticated microscopes and image analysis techniques. Due to its high-contrast, high-specificity output, many assays rely on confocal fluorescence microscopy, genetic methods, or antibody staining techniques. These approaches often limit the ability to measure quantitatively dynamic activity such as intracellular transport and growth. In this work, we describe a method for label-free live-cell cell imaging with antibody staining specificity by estimating the associated fluorescence signals via quantitative phase imaging and deep convolutional neural networks. This computationally inferred fluorescence image is then used to generate a semantic segmentation map, annotating subcellular compartments of live unlabeled neural cultures. These synthetic fluorescence maps were further applied to study the time-lapse development of hippocampal neurons, highlighting the relationships between the cellular dry mass production and the dynamic transport activity within the nucleus and neurites. Our implementation provides a high-throughput strategy to analyze neural network arborization dynamically, with high specificity and without the typical phototoxicity and photobleaching limitations associated with fluorescent markers.

Evaluation of in-situ fatty acid extraction protocols for the analysis of staphylococcal cell membrane associated fatty acids by gas chromatography

The composition and integrity of the bacterial cytoplasmic membrane is critical to the survival of staphylococci in dynamic environments and it is important to investigate how the cell membrane responds to changes in the environmental conditions. The staphylococcal membrane differs from eukaryotic and many other bacterial cell membranes by having a high abundance of branch fatty acids and relatively few unsaturated fatty acids. The range of available methods for extraction and efficient analyses of staphylococcal fatty acids was initially appraised to identify the best potential procedures for appraisal. Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 29213) was grown under optimal conditions to generate a cell biomass to compare the efficiencies of three approaches to extract and prepare methyl esters of the membrane fatty acids: (1) acidic direct transesterification of lipids, (2) modified basic direct transesterification of membrane lipids with adjusted reaction times and temperatures, and (3) base catalysed hydrolysis followed by acid catalysed esterification in two separate chemical reactions (MIDI process). All methods were able to extract fatty acids from the cell mass effectively where these lipids represented approximately 5% of the cellular dry mass. The acidic transesterification method had the least number of steps, the lowest coefficient of variation at 6.7% and good resistance to tolerating water. Basic transesterification was the least accurate method showing the highest coefficient of variation (26%). The MIDI method showed good recoveries, but had twice the number of steps and a coefficient of variation of 16%. It was also found that there was no need to use an anti-oxidant such as BHT for the protection of polyunsaturated fatty acids when the GC–MS injection liner was clean. It was concluded that the acidic transesterification procedures formed the most efficient and reproducible method for the analyses of staphylococcal membrane fatty acids.

Effect of cilofungin on the growth and metabolite production in spirulinasp and chlorellasp

A novel strategy of using an antifungal cilofungin to alter the algal metabolics for increased production of biomass, fat, protein and other metabolites were inferred. The effect of varying concentrations of cilofungin (0.02% to 0.1%) on unialgal cultures of Spirulina sp. and Chlorella sp. isolated from ennore estuary, Chennai, an highly polluted coastal ecosystem in India was investigated. Various parameters like cellular growth, dry mass, carbohydrates, proteins, fatty acids, chlorophyll and carotenoids were assessed with respect to time. At a given cilofungin concentration of 0.08% in Spirulina sp and 0.06% in Chlorella sp, an increase in protein content from 421mg/g to 655mg/g and 430mg/g to 646 mg/g of biomass, an increase in fat content from 57mg/g to 74 mg/g and 75 mg/g to 90mg/g of biomass was obtained in comparison to their mother cultures with a simultaneous increase in their biomass concentration from 1.09g/l to 1.5g/l and 1 g/l to 1.23g/l respectively, whereas the carbohydrates were gradually decreased in cilofungin treated cultures. This study thus analyse the effect of cilofungin in algae, inhibiting the glucan production and inducing the alternate metabolic pathways in Spirulina sp and Chlorella sp. for lipid and protein accumulation.

Inflating bacterial cells by increased protein synthesis.

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7- to 8-fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3- to 4-fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ∼8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.

Angle-resolved light scattering of individual rod-shaped bacteria based on Fourier transform light scattering.

Two-dimensional angle-resolved light scattering maps of individual rod-shaped bacteria are measured at the single-cell level. Using quantitative phase imaging and Fourier transform light scattering techniques, the light scattering patterns of individual bacteria in four rod-shaped species (Bacillus subtilis, Lactobacillus casei, Synechococcus elongatus, and Escherichia coli) are measured with unprecedented sensitivity in a broad angular range from −70° to 70°. The measured light scattering patterns are analyzed along the two principal axes of rod-shaped bacteria in order to systematically investigate the species-specific characteristics of anisotropic light scattering. In addition, the cellular dry mass of individual bacteria is calculated and used to demonstrate that the cell-to-cell variations in light scattering within bacterial species is related to the cellular dry mass and growth.

Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions

Even a casual pass through the great halls of mammals in the world’s natural history museums highlights the wide diversity of skeletal proportions that allow us to distinguish between species even when reduced to their calcified components. Similarly each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation1–7. Despite this recognized importance, the mechanisms of chondrocyte volume enlargement have remained a mystery8–11. Here we use quantitative phase microscopy12 to show that chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types13, this stands as a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through an Insulin-like Growth Factor-dependent mechanism. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify, and establish a volume set point.

Optical Quantification of Cellular Mass, Volume, and Density of Circulating Tumor Cells Identified in an Ovarian Cancer Patient

Clinical studies have demonstrated that circulating tumor cells (CTCs) are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal, and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD)-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC) compared to 18.7 ± 0.6 pg (leukocytes), p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC) compared to 230.9 ± 78.5 fL (leukocyte), p < 0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC) compared to 0.085 ± 0.004 pg/fL (leukocyte), p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.

Imaging the cellular response to transient shear stress using stroboscopic digital holography

We use stroboscopic quantitative phase microscopy to study cell deformation and the response to cavitation bubbles and transient shear stress resulting from laser-induced breakdown of an optically trapped nanoparticle. A bi-directional transient displacement of cytoplasm is observed during expansion and collapse of the cavitation bubble. In some cases, cell deformation is only observable at the microsecond time scale without any permanent change in cell shape or optical thickness. On a time scale of seconds, the cellular response to shear stress and cytoplasm deformation typically leads to retraction of the cellular edge most exposed to the flow, rounding of the cell body and, in some cases, loss of cellular dry mass. These results give a new insight into the cellular response to cavitation induced shear stress and related plasma membrane permeabilization. This study also demonstrates that laser-induced breakdown of a nanoparticle offers localized cavitation, which interacts with a single cell but without causing cell lysis.

Optical imaging of cell mass and growth dynamics

Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or "dry mass" of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells.

Microdiesel: Escherichia coli engineered for fuel production

Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l(-1) and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.

In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized. The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the ( R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified. The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P. oleovorans.

Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn 5 kanamycin resistance gene block ( neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30–50% less PHA than the P. putida wild type.

Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria

Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3-hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4–10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the α-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acid-coenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate.

Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid byRhodococcus ruberNCIMB 40126 and related bacteria

Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3-hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4–10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the α-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acid-coenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate.

Diverse Mechanisms for Regulating Ribosomal Protein Synthesis in Escherichia coli

Publisher Summary This chapter describes the current knowledge of the organization and regulation of ribosomal genes, emphasizing recent work elucidating the molecular mechanisms regulating ribosomal protein synthesis and illustrating the diversity of these mechanisms. In E. coli, the small ribosomal subunit contains one rRNA molecule (16 S) and 21 proteins. The large subunit harbors two rRNA molecules (23 S and 5 S) and 34 proteins. All of the genes for these ribosomal components have been mapped on the E . coli chromosome. Furthermore, all but three of the r-protein genes have been sequenced. Because proteins are the most abundant cellular macromolecules, cell growth requires that a large fraction of the cell mass be devoted to the protein synthesis machinery— namely, the ribosomes and their ancillary factors. For example, in rapidly growing bacteria, ribosomes account for as much as 50% of cellular dry mass. Because ribosome formation commands such a large fraction of cellular resources, even minor adjustments in the rate of ribosome synthesis can have a major impact on the economy of the cell.

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.

Intracellular ion concentrations and cell volume during cholinergic stimulation of eccrine secretory coil cells

Methacholine (MCh)-induced changes in intracellular concentrations of Na, K, and Cl [( Na]i, [K]i, and [Cl]i, respectively) and in cellular dry mass (a measure of cell shrinkage) were examined in isolated monkey eccrine sweat secretory coils by electron probe X-ray microanalysis using the peripheral standard method. To further confirm the occurrence of cell shrinkage during MCh stimulation, the change in cell volume of dissociated clear and dark cells were directly determined under a light microscope equipped with differential interference contrast (DIC) optics. X-ray microanalysis revealed a biphasic increase in cellular dry mass in clear cells during continuous MCh stimulation; an initial increase of dry mass to 158% (of control) followed by a plateau at 140%, which correspond to the decrease in cell volume of 37 and 29%, respectively. The latter agrees with the MCh-induced cell shrinkage of 29% in dissociated clear cells. The MCh-induced increase in dry mass in myoepithelial cells was less than half that of clear cells. During the steady state of MCh stimulation, both [K+]i and [Cl]i of clear cells decreased by about 45%, whereas [Na]i increased in such a way to maintain the sum of [Na] i + [K]i constant. There was a small (12-15 mM) increase in [Na]i and a decrease in [K]i in myoepithelial cells during stimulation with MCh. Dissociated dark cells failed to significantly shrink during MCh stimulation. The decrease in [Cl]i in the face of constant [Na]i + [K]i suggests the accumulation of unknown anion(s) inside the clear cell during MCh stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)