Any study regarding the influence of physiological, pathological, or pharmacological/toxicological factors on cellular ion homeostasis requires knowledge of multiple parameters including cytoplasmic ion concentrations, ion fluxes, and subcellular elemental distribution so that an accurate assessment of the state of ion homeostasis can be obtained. These multiple parameters are often obtained by using different types of microscopy and microanalysis. For example, cytoplasmic ion concentrations are measured by fluorescence digital imaging microscopy using various ion indicators, while cellular and subcellular element contents are determined by electron probe X-ray microanalysis (EPXMA) digital imaging. For correlating information from various microscopies, it would be advantageous to have one software package and image format that operates on the same computer platform. Here, we describe the modification of an existing software (ImagNSpect) developed for EPXMA digital imaging for use in analyzing fluorescence digital images.
Fluorescence images (490 and 440 nm excitation, 540 nm emission) of BCECF-loaded cultured rat hepatocytes (Fig. 1a, b) were obtained on an inverted fluorescence microscope (Carl Zeiss, Inc., Thorn wood, NY) equipped with a rotating excitation filter wheel, a CCD camera (Photometries, Ltd., Tucson, AZ), a controller box, and a Macintosh Quadra 800 computer (Apple Computer Corp., Cupertino, CA).