A promising microscopic technique for detecting Schistosoma mansoni eggs.

Schistosomiasis is one of the most devastating neglected tropical diseases, affecting over 250 million people worldwide and contributing to approximately 280,000 deaths annually. Microscopic detection of Schistosoma mansoni eggs in fecal samples using the Kato-Katz method remains the diagnostic standard. However, its speed and detection efficiency are limited.

A guide to transport-of-intensity equation (TIE) imaging for biologists.

The measurement of dry cell mass (often referred to as "protein") under the microscope can be accomplished using a quantitative phase imaging technique known as Transport of Intensity Equation (TIE) microscopy. This method requires no specialized equipment, relying instead on two slightly defocused brightfield images acquired with a standard optical microscope. The images are processed by the TIE equation to convert the gradient of intensity into phase shifts and ultimately a distribution of protein mass.

Label-Free Quantification of Protein Density in Living Cells.

Intracellular water content, W, and protein concentration, P, are essential characteristics of living cells. Healthy cells maintain them within a narrow range, but often become dehydrated under severe stress; moreover, persistent loss of water (an increase in P) can lead to apoptotic death. It is very likely that protein concentration affects cellular metabolism and signaling through macromolecular crowding (MC) effects, to which P is directly related, but much remains unknown in this area.

Delayed vacuolation in mammalian cells caused by hypotonicity and ion loss.

Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter.

Measurement of protein concentration in bacteria and small organelles under a light transmission microscope.

Protein concentration (PC) is an essential characteristic of cells and organelles; it determines the extent of macromolecular crowding effects and serves as a sensitive indicator of cellular health. A simple and direct way to quantify PC is provided by brightfield-based transport-of-intensity equation (TIE) imaging combined with volume measurements. However, since TIE is based on geometric optics, its applicability to micrometer-sized particles is not clear.

Selective Visualization of Live Intestinal Parasites in Stool Specimens Without Purification.

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 problem.

Stability of Intracellular Protein Concentration under Extreme Osmotic Challenge.

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 (PC) in HeLa and 3T3 cells for up to 48 h. Long-term PC remained stable in solutions with osmolarities ranging from one-third to almost twice the normal.

Studying cell volume beyond cell volume.

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 the same cells. The reason for combining cell volume with dry mass is that the ratio of the two-dry mass concentration (MC)-is an important and insufficiently utilized biological parameter.

Experimental test of the geometric model of image formation in bright-field microscopy.

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 experimentally. In this paper, we applied simple ray tracing to compute an image of a glass cylinder at various positions of the objective and to compare it to the experiment.

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else.

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.

A Reverse-Osmosis Model of Apoptotic Shrinkage.

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 another mechanism of apoptotic dehydration based on cytoskeletal compression.

Comparison of cell volume measurements by fluorescence and absorption exclusion microscopy.

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 absorbing dye, resulting in an increased intensity of transmission image.

Evidence for macromolecular crowding as a direct apoptotic stimulus.

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 volume could be restored and further reduced by application of sucrose.

Cell Volume Measurements by Optical Transmission Microscopy.

Cell volume is an important parameter in studying cell adaptation to anisosmotic stress, activation of monovalent ion channels, and cell death. This article describes a method for measurement of the volumes of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell-impermeant dye, Acid Blue 9.

Calibration and characterization of intracellular Asante Potassium Green probes, APG-2 and APG-4.

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular and extracellular concentrations of the measured ion must be made equal. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium.

Intracellular Macromolecules in Cell Volume Control and Methods of Their Quantification.

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.

Staurosporine-induced apoptotic water loss is cell- and attachment-specific.

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.

Intermolecular G-Quadruplex Induces Hyaluronic Acid-DNA Superpolymers Causing Cancer Cell Swelling, Blebbing, and Death.

Over the past decade, nanomedicine has gained considerable attraction 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 derivatized a thiolated conjugate of the naturally occurring glycosaminoglycan polymer hyaluronic acid (HASH) with short G-rich DNA (HASH-DNA) that can form an intermolecular noncanonical GQ structure.

Bright-Field Microscopy of Transparent Objects: A Ray Tracing Approach.

The 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 the 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 focal plane caused by the object.

A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

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 by FSC problematic.

Methods for cell volume measurement.

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-slice imaging, intracellular volume markers, displacement of extracellular solution, quantitative phase imaging, radioactive methods, and some others. Suitability of these methods to some typical samples and applications is discussed.

Calibrated brightfield-based imaging for measuring intracellular protein concentration.

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 biological cells.

Unidirectional Flux Balance of Monovalent Ions in Cells with Na/Na and Li/Na Exchange: Experimental and Computational Studies on Lymphoid U937 Cells.

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.