Automated Measurements of Fluorescence in Spatially Defined Regions of Pollen Tubes

Pollen tubes are a widely used model for the extension and development of polarized cells. Their relatively standard shape simplifies automated identification and image analysis of subregions. The key is reliable identification of the cell plasma membrane and the pollen tube tip. This chapter describes how regions of interest can be created and used to assess distributions in tubular cells using two ImageJ macros. The first macro enables efficient and precise identification of the pollen tube plasma membrane and tip, based on a few imprecisely marked points. Subsequent steps are automated, with users prompted to confirm their accuracy, a balance between automation and supervision. The plasma membrane and the tip are the bases of five distance maps: three starting from the tip, one from the plasma membrane, and one from the pollen tube midline. Measurements of fluorescence intensity and generalized polarization gradients are then made from the tip along the plasma membrane. The second macro uses the distance maps to measure fluorescence intensities and generalized polarization from user-specified regions derived from a single or a combination of the five distance maps. Highly reproducible measurements are achieved by automation with minimal user interaction, while every analysis step is recorded for complete traceability.