Application of fluorescence microscopy and image analysis for quantifying dynamics of maize pollen shed

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Abstract
The objective of this study was to develop a simple, rapid and accurate technique to quantify maize (Zea mays L.) pollen shed under field conditions, capitalizing on the capacity of pollen to fluoresce and recent improvements in microscopic methods for acquiring, processing, and analyzing fluorescence signals from biological systems. Pollen shed naturally by the tassels was captured daily on passive pollen traps placed at apical ear level. Fluorescence microscopy was used to generate digital images of the trapped pollen, and pollen density per unit area was counted using commercial imaging software. Visual confirmation of fluorescing pollen grains indicated high measurement accuracy (r2 = 0.99) for the entire range of pollen shed densities typically encountered in the field. Pollen was randomly distributed across the surface of the pollen trap, and six to eight images per trap provided greater than 95% confidence for the mean trap value. The entire process of sample preparation, image capture, and image counting required less than 6 min per trap. The accuracy and ease of use of this technique make it ideal for characterizing the pattern of maize pollen production and dispersal under field conditions.