L G Alexopoulos | PubMed Health

PubMed2002

A method for quantifying cell size from differential interference contrast images: validation and application to osmotically stressed chondrocytes.

An automatic image analysis method was developed to determine the shape and size of spheroidal cells from a time series of differential interference contrast (DIC) images. The program incorporates an edge detection algorithm and dynamic programming for edge linking. To assess the accuracy and working range of the method, results from DIC images of different focal planes and resolutions were compared to confocal images in which the cell membrane was fluorescently labelled. The results indicate that a 1-microm focal drift from the in-focus plane can lead to an overestimation of cell volume up to 14.1%, mostly due to shadowing effects of DIC microscopy. DIC images allow for accurate measurements when the focal plane lies in a zone slightly above the centre of a spherical cell. In this range the method performs with 1.9% overall volume error without taking into account the error introduced by the representation of the cell as a sphere. As a test case, the method was applied to quantify volume changes due to acute changes of osmotic stress.

Animals↗

PubMed2001

Hyper-osmotic stress induces volume change and calcium transients in chondrocytes by transmembrane, phospholipid, and G-protein pathways.

Mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways. One of the early events implicated in regulatory volume changes and mechanotransduction is an increase of intracellular calcium ion ([Ca(2+)](i)). In this study, we tested the hypothesis that osmotic stress initiates intracellular Ca(2+) signaling in chondrocytes. Using laser scanning microscopy and digital image processing, [Ca(2+)](i) and cell volume were monitored in chondrocytes exposed to hyper-osmotic solutions. Control experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as transient increases in [Ca(2+)](i). The initial peak in [Ca(2+)](i) was generally followed by decaying oscillations. Pretreatment with gadolinium, a non-specific blocker of mechanosensitive ion channels, inhibited this [Ca(2+)](i) increase. Calcium-free media eliminated [Ca(2+)](i) increases in all cases. Pretreatment with U73122, thapsigargin, or heparin (blockers of the inositol phosphate pathway), or pertussis toxin (a blocker of G-proteins) significantly decreased the percentage of cells responding to osmotic stress and nearly abolished all oscillations. Cell volume decreased with hyper-osmotic stress and recovered towards baseline levels throughout the duration of the control experiments. The peak volume change with 550 mOsm osmotic stress, as well as the percent recovery of cell volume, was dependent on [Ca(2+)](i.) These findings indicate that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in [Ca(2+)](i).

Animals↗