Improved cytoplasmic pH measurements in SNARF-1 stained plant cells by image processing

Cytoplasmic pH has long been considered to act as a secondary messenger of various cellular responses by affecting the ionization state of proteins. In plant biology, cytoplasmic pH has traditionally been measured, especially in guard cells and as a response to plant microorganism interactions, with pH-sensitive microelectrodes. More recently, the development of fluorescent pH markers, such as BCECF and SNARF-1, has allowed us to monitor cytoplasmic pH without the need for electrophysiological equipment. However, because of vacuolar structures that occupy a large volume of plant cells, simple measurements of fluorescent intensities are insufficient to provide precise cytoplasmic pH values. In this addendum, we describe our improved method to monitor cytoplasmic pH in plant cells stained by SNARF-1 by image processing using a noise-reducing filter after determination of an optimal ROI size. In addition, further developments for automated region extraction are proposed.