Single-cell volume estimation by three-dimensional wide-field microscopy applied to astroglial primary cultures

Astrocytes, which constitute a prominent part of the number and volume of b rain cells, have a high capacity for controlling their volume, and astrocyt ic swelling is associated with a number of pathological states affecting th e CNS. In order to understand the mechanisms for regulating cell volume in astrocytes better, it is of utmost importance to develop technical instrume ntation and analysis methods capable of detecting and characterizing dynami c cell shape changes in a quantitative and robust way. For this purpose, a new method was developed to quantify changes in cell volume at the single-c ell level. This method is based on three-dimensional (3D) fluorescence imag ing obtained by optical sectioning. An automated image acquisition system w as developed for the collection of two-dimensional (2D) microscopic images. A deblurring algorithm was implemented in order to restore the originally unfocused image content. Advanced image analysis techniques were applied fo r accurate and automated determination of cell volume. The sensitivity and reproducibility of the method was evaluated by using fluorescent beads. The techniques were applied to fura-2-labeled astroglial cells in primary cult ure exposed to hypo- or hyperosmotic stress. The results show that this met hod is valuable for determining volume changes in cells or parts thereof. ( C) 1999 Elsevier Science B.V. All rights reserved.