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 brain cells, have a high capacity for controlling their volume, and astrocytic swelling is associated with a number of pathological states affecting the CNS. In order to understand the mechanisms for regulating cell volume in astrocytes better, it is of utmost importance to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic 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-cell level. This method is based on three-dimensional (3D) fluorescence imaging obtained by optical sectioning. An automated image acquisition system was 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 for 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 culture exposed to hypo- or hyperosmotic stress. The results show that this method is valuable for determining volume changes in cells or parts thereof.