Michael Model

Kent State University | KSU · Department of Biological Sciences

Purpose: Microscopic observation of live parasites in the stool is an important diagnostic tool in human and veterinary medicine. Because of the presence of large amounts of contaminating organic matter, microscopic analysis must be preceded by time-consuming pre-purification steps. Transmission-through-dye (TTD) optical microscopy obviates this p...

Cell volume (CV) regulation is typically studied in short-term experiments to avoid complications resulting from cell growth and division. By combining quantitative phase imaging (by transport-of-intensity equation) with CV measurements (by the exclusion of an external absorbing dye), we were able to monitor the intracellular protein concentration...

en In the geometric optics approximation, an image formed by an objective lens replicates the distribution of intensity at the front focal plane of the objective. Although this fact represents a fundamental optical principle, its application to analysis of bright‐field microscopic images was developed only recently and has not been tested experimen...

High density of intracellular macromolecules creates a special condition known as macromolecular crowding (MC). One well-established consequence of MC is that only a slight change in the concentration of macromolecules (e.g., proteins) results in a shift of chemical equilibria towards the formation of macromolecular complexes and oligomers. This su...

The first part of the paper describes two simple microscopic techniques that we use in our laboratory. One measures cell volumes in adherent cultures and the other measures cell dry mass; both measurements are done on the same instrument (a standard bright-field transmission microscope with only one or two narrow-band color filters added) and on th...

The standard theory of apoptotic volume decrease (AVD) posits activation of potassium and/or chloride channels, causing an efflux of ions and osmotic loss of water. However, in view of the multitude of possible channels that are known to support apoptosis, a model based on specific signaling to a channel presents certain problems. We propose anothe...

Although research and new technologies have introduced different ways of observing microorganisms, including scanning and electron microscopy, these methods are expensive and require equipment that is typically not found in a middle school classroom. The transmission-through-dye technique (TTD; Gregg et al., 2010), a new optical microscopy method t...

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial...

There are two light microscopic methods for cell volume measurement based on volume exclusion. One method, sometimes referred to as FLEX, utilises negative staining by an external fluorescent dye, and cell volume is found from attenuation of fluorescence under a wide‐field microscope. The other method (TTD) is based on exclusion of an external abso...

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this s...

Over the past decade, nanomedicine has gained considerable traction through its relevance, for example in “smart” delivery, thus creating platforms for novel treatments. Here we report a natural polymer-DNA conjugate that undergoes self-assembly in a K+ dependent fashion to form a G-quadruplex (GQ) and generate superpolymeric structures. We derivat...

The traditional theories of cell volume regulation focus on monovalent ions and small organic osmolytes. The main subject of this review is macromolecular content of the cell and its role in cell volume. We start by reviewing general information about cellular macromolecules and present some quantitative relationships. Next, we review a wide range...

A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD...

Necrotic cells are known to develop characteristic membrane blebs. We measured protein concentration within necrotic blebs and found that it can be reduced by as much as twenty-fold compared to the main cell body (CB). These results raise two questions: 1. Why do proteins vacate the bleb? 2. How can osmotic equilibrium be maintained between the ble...

Volume is an essential characteristic of a cell, and this review describes the main methods of its measurement that have been used in the past several decades. The discussed methods include various implementations of light scattering, estimates based on one or two cell dimensions, surface scanning, fluorescence confocal and transmission slice-by-sl...

Intracellular protein concentration is an essential cell characteristic, which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on...

Formation of a bright-field microscopic image of a transparent phase object is described in terms of elementary geometrical optics. Our approach is based on the premise that image replicates the intensity distribution (real or virtual) at the front focal plane of the objective. The task is therefore reduced to finding the change in intensity at the...

Monovalent ion traffic across the cell membrane occurs via various pathways. Evaluation of individual fluxes in whole cell is hampered by their strong interdependence. This difficulty can be overcome by computational analysis of the whole cell flux balance. However, the previous computational studies disregarded ion movement of the self-exchange ty...

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium,...

Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medi...

Most cells carry a negative electric charge. It produces a potential difference across the membrane, which regulates voltage-sensitive ion transport and ATP synthesis in mitochondria. The negative charge comes partly from an excess of negative ions in the cell interior (Donnan potential) and partly from ionized groups on the membrane (surface poten...

Many vital processes in animal cells depend on monovalent ion transport across the plasma membrane via specific pathways. Their operation is described by a set of nonlinear and transcendental equations that cannot be solved analytically. Previous computations had been optimized for certain cell types and included parameters whose experimental deter...

Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat-field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide an...

Cell shrinkage and dehydration are essential characteristics of apoptosis, and the loss of as much as half of the initial cell volume is not uncommon. This phenomenon is usually explained by the efflux of potassium and chloride ions. We reexamine this hypothesis based on the available data for ion concentrations and taking into account the requirem...

Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport-of-intensity equa...

of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

An optical measurement system for obtaining information such as the absorption coefficient of a light-absorbing liquid, or a surface profile of an object immersed in a light-absorbing liquid having a known absorption coefficient. The system includes a light source that transmits light through the liquid, a detector that records an image of the ligh...

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential featu...

Background/Aims: Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relations...

Shape and size are among the most basic and obvious characteristics of a cell (or of any physical object, for that matter). When a cell is observed through a microscope, one only sees its projection onto the image plane. Rather paradoxically, there are no easy techniques to visualize and measure cell's third dimension—thickness. For example, confoc...

Bacterial phenotypes result from responses to environmental conditions under which these organisms grow; reduced gravity has been demonstrated in many studies as an environmental condition that profoundly influences microorganisms. In this study, we focused on low-shear stress, modeled reduced gravity (MRG) conditions and examined, for Escherichia...

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.

Conventional light microscopy techniques are poorly suited for imaging the vertical cell dimension. This can be accomplished using transmission-through-dye (TTD) imaging, in which cell thickness is directly converted into image intensity in the presence of extracellular dye with strong absorption. We have previously described applications of TTD to...

Refractive index mismatch between the specimen and the objective immersion oil results in spherical aberration, which causes distortion and spreading of the point spread function, as well as incorrect readings of the axial coordinates. These effects have to be taken into account when performing three-dimensional restoration of wide-field fluorescen...

Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cell...

Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid laye...

We propose a method to image the surface topography of transparent objects. The space between the object and the opposite closely positioned surface (such as a cover glass or a slide) is filled with a strongly absorbing dye. The contrast is generated by recording a transmission image at a wavelength where the dye absorbs. Since the transmitted inte...

The axial spread function is a useful tool for evaluation of a confocal microscope. It can be obtained experimentally by scanning a uniform fluorescent layer whose thickness is significantly below the resolution limit. Previous researchers have created thin fluorescent films by chemical synthesis. We show here that concentrated fluorescent dyes wit...

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emissi...

Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated dye solution placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photob...

Numerous applications of fluorescence microscopy require quantitation of signal intensity in reproducible units. Two problems must be overcome to achieve this goal. First, due to various instrumental factors, the same sample imaged on two microscopes or even on the same microscope at different times may produce highly divergent readings. Second, be...

Fluorescence microscopy can offer unique advantages for biomaterials characterization. Like spectroscopy or radioactivity, it can be used to quantify specific binding to surfaces, but it can also assess surface homogeneity at the micron scale or detect protein aggregation. To fully utilize the potential of this technique, there must be a way to cal...

In detecting receptor antagonists or enzyme inhibitors, there are three parameters that often affect the outcome in a predictable quantitative manner: concentrations of the receptors (enzyme), labeled ligand (substrate), and antagonist (inhibitor). The usual goal of assay optimization is to maximize the ability of the assay to detect low concentrat...

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses have been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. Thes...

We investigated the relationship between the respiratory burst in neutrophils and the membrane distribution of the IgG receptor, FcγRIII.FcγRIII receptors were labeled with a fluoresceinated antibody that does not block binding of immune complexes.The respiratory burst was detected using covalently bound rosamine stain previously described for flow...

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses has been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These...

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sen...

Neutrophils stimulated with a low concentration of chemotactic peptide N-formyl-met-leu-phe respond with a brief pulse of actin polymerization and a persistent change in morphology from round to polarized. The recovery of the actin polymerization response is unlikely to be due to usual receptor desensitization mechanisms. We hypothesized that cell...

The rate of binding of a ligand to receptors on the cell surface can be diffusion limited. We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport. We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient. A moderately transport-limited kine...

Many chemoattractant-activated responses in neutrophils show transient kinetics, suggesting that rapid desensitization occurs during the time course of the response. We found that desensitization of the actin polymerization response to N-formyl peptides is, in a large part, due to inhibition by adenosine released from cells to the medium and deplet...

Regulation of cellular responses to stimulation is one of the major areas in contemporary biology. I studied regulation of the actin polymerization response in neutrophils stimulated by chemotactic peptides. Actin polymerization in neutrophils, as well as in many other cell types, is essential for polarization, motility, chemotaxis, and phagocytosi...

A simplified procedure is developed for estimation of glutathione peroxidase activity involving glutathione reductase in the conjugated reaction. The procedure developed enabled to use commonly employed spectrophotometers and to decrease distinctly the glutathione reductase consumption. Isolation of glutathione reductase from yeast is described.