Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.     

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Coherent phase microscopy: a new method of identification of intracellular structures based on their optical and morphometric parameters

A new method for the identification of intracellular structures of a living cell and obtaining the quantitative parameters characterizing these structures by means of coherent phase microscopy is proposed. The method is based on the analysis of the histogram of a cell phase image and its decomposition by phase height levels. In the spherical and cylindrical approximation of the cell, the method makes it possible to separate the contributions of phase-contrast intracellular structures to the integral refractive index of the whole cell. The calculation of refractive indices of intracellular structures is illustrated on a two-component model of a spherical cell. The possibility of determining the refractive indices of cellular organelles was shown by an example of cyanobacterium Anabaena variabilis ATCC 29413 cells and Cancer mammae breast cancer cells. Three most contrast cellular structures in the phase images of the cells were identified, and their refractive indices were determined. For Anabaena variabilis cells, these structures were the cell wall ( = 1.39), tylakoids ( = 1.47), and the nucleoid ( = 1.58); for breast cancer cells, these were the cytoplasm ( = 1.34), the nucleus ( = 1.36), and the nucleolus ( = 1.44). Using this method is possible to identify phase-contrast intracellular structures and calculate their refractive indices. This enables one to follow morphofunctional changes of not only the whole cell but also its individual organelles.     

IDENTIFICATION OF A CYCLIC AMP-DEPENDENT PROTEIN KINASE IN THE DINOFLAGELLATE AMPHIDINIUM OPERCULATUM

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

MEASUREMENT OF MICROALGAL CELL VOLUME BY FLOW CYTOMETRY

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

Cellular composition of the cyclic corpus luteum of the cow

The cellular composition of CL from 6 cows on approximately Day 12 of the oestrous cycle, after synchronization with cloprostenol, was studied by ultrastructural morphometry. Point-count measurements of volume density (mean +/- s.d.) showed that large luteal cells occupied 40.2 +/- 7.0% of the luteal tissue, and small luteal cells 27.7 +/- 6.3%. Of the total of 393.4 +/- 52.0 x 10(3) cells per mm3 of luteal tissue, large luteal cells made up only 3.5% and small luteal cells 26.7%, a ratio of 1:7.6. Endothelial cells/pericytes, at 52.3%, were the most numerous cell type. The mean volume per large luteal cell was 29.6 +/- 6.3 x 10(3) microns 3, while that of small luteal cells was 2.7 +/- 0.4 x 10(3) microns 3. In spherical form, these volumes would represent mean diameters of 38.4 microns and 17.2 microns respectively, and are consistent with published measurements on dispersed luteal cells. However, the values for cell numbers are much higher than published values based on luteal tissue dispersion, suggesting that dispersion may result in substantial and possibly selective losses of luteal cells.     

Methods of measuring volume changes in erythrocytes under hypoosmotic stress: a comparison

OBJECTIVE: To compare methods of analyzing changes in red blood cell volume when exposed to osmotic challenge. STUDY DESIGN: The sensitivity of different methods was tested using fish erythrocytes under hypoosmotic stress (80% and 60% saline): hematocrit ratio, Hcto(exp)/ Hcto(ctr); cellular hemoglobin mean concentration; optical density (OD400); water content; surface area estimation; mean corpuscular volume (MCV); and mean cellular hemoglobin (MCH). RESULTS: Cells exposed to both solutions demonstrated a volume increase via hematocrit ratio of 16 +/- 2% and 30 +/- 3% (p < 0.001) when exposed to 80% and 60% saline, respectively. Cellular volume estimation by OD400 revealed a change in volume only at 60% saline (26% increase). Surface area estimation suggested a difference in volume at 60% saline. MCV revealed differences in volume at < 60% saline exposure with a 33% increase and indicated a 19% increase at 80% saline. MCH showed an increase of 48% with exposure to 80% saline. CONCLUSION: The results reveal that Hcto(ctr)/Hcto(exp), MCV, OD400 and MCH yield significant volume changes and that Hcto(ctr)/Hcto(exp) is the most sensitive assay.     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.     

Influence of intracellular trehalose concentration and pre-freeze cell volume on the cryosurvival of rapidly frozen human erythrocytes

Significant interest exists in the application of trehalose, which has low permeability to the phospholipid bilayer, as a non-toxic intracellular cryopreservative for mammalian cells. Introduction of between 8 ± 3 mM and 266 ± 22 mM trehalose into human erythrocytes using the membrane permeabilizing polymer PP-50 allowed investigation of the relationship between intracellular trehalose concentration, pre-freeze cell volume, and cryosurvival. Cellular cryosurvival increased approximately linearly with pre-freeze cell volume up to the normal volume of fresh cells; diminished cell survival correlated with subnormal pre-freeze cell volume in some cases even at >100 mM intracellular trehalose concentration. Uptake of >200 mM trehalose in cells with near-normal cell volume facilitated enhancement of cellular cryosurvival by up to 15 ± 5%.     

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.     

Taurine content of isolated rat alveolar type I cells.

1. Rat alveolar type I cells were isolated by enzymatic digestion and purified by centrifugal elutriation and specific surface adsorption. 2. The identity of the harvested cells was confirmed using electronic cell sizing and transmission electron microscopy. 3. Purified cell preparations contained 4.6 +/- 2.3 x 10(6) type I cells/rat lung with a purity of 79 +/- 3%. 4. Isolated type I cells exhibited the following characteristics: mean cell volume = 716 +/- 48 microns 3; diameter = 11.1 +/- 0.7 microns; and cell water content = 0.50 +/- 0.03 microliter/10(6) cells. 5. Taurine content of these alveolar type I cells was measured by HPLC. 6. The intracellular taurine concentration of type I cells was 0.14 +/- 0.07 mM, a value close to that of plasma (0.1 mM).     

Mechanism of erythrocyte cryohemolysis, induced by cationic amphipaths: synergism of induction of the "discocyte-stomatocyte III" transition due to chlorpromazine and medium tonicity]

It is determined that chlorpromazine, a cationic amphipath usually protecting erythrocytes under conditions of hypertonic cryohemolysis is an efficient inductor of the cell lysis in case of cooling in media with tonicity close to the physiological (the isotonic cryohemolysis). Both chlorpromazine and tonicity of the medium influence the alterations in the state of cells, which is confirmed by synergy of the "discocyte-stomatocyte III" transition induction. The above process may be considered as a critical stage of structural modification of erythrocytes. Transition through this stage coincides with appearance of sensitivity to cooling in cells.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5-2.0% w/v) concentrations of Dextran T500. At concentrations of 5-7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

Monovalent cation metabolism and cytopathic effects of poliovirus-infected HeLa cells.

To better understand the significance of 22Na+ accumulation by poliovirus-infected HeLa cells (C. N. Nair, J. W. Stowers, and B. Singfield, J. Virol. 31:184, 1979), measurements of cellular Na+, K+, and Cl- contents, volume, and density were carried out at intervals after infection. In addition, the rates of 22Na+ washout from infected and control cells were determined. Starting at around 3 h postinfection, the Na+ content of infected cells increased, whereas the K+ content decreased progressively, resulting in a net loss in the monovalent cation content decreased progressively, resulting in a net loss in the monovalent cation content per cell. The loss in cellular chloride content exceeded that in monovalent cation content. The kinetics of 22Na+ washout from infected and control cells revealed the presence of an extra Na+ compartment in infected cells. A net loss in the monovalent cation activity of infected cells was indicated by the loss of cell water as reflected in a decrease in cell volume and an increase in cell density. In spite of a net loss in monovalent cation content per cell, Na+ accumulation coupled with cell shrinkage resulted in substantial increases in the concentrations of not only Na+ but also K+. The results suggested a possible role for tonicity change in the morphological lesions of poliovirus cytotoxicity.     

Unraveling Cell Processes: Interference Imaging Interwoven with Data Analysis

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1–0.6 Hz) result from plasma membrane processes and that higher frequency variations (20–26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties.     

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.     

Evaluation of an electrochemical model of erythrocyte pH buffering using 31P nuclear magnetic resonance data

When erythrocytes are suspended in a solution of known composition the resultant values of such basic cell parameters as volume and pH are difficult to predict. To facilitate such predictions, we developed a mathematical model describing the passive transmembrane distribution of permeant species; three simultaneous equations were produced. Certain essential data required for the model were determined experimentally; these included the pH dependence of the charge on the hemoglobin molecule and the variation of the osmotic coefficient of hemoglobin with cell volume. Finally, cells were added to various solutions, and then titrated to produce a wide pH range (pH 6-8). We measured the resultant cell volume, cellular and extracellular pH using both conventional and 31P NMR methods. The expected equilibrium values of these electrochemical parameters were also calculated by solving (numerically) the three model equations. The accuracy of the model simulations was evaluated by direct comparison of calculated and experimentally determined values.