Pollen grains represent the male gametes of seed plants and their viability is critical for sexual reproduction in the plant life cycle. Palynology and viability studies have traditionally been used to address a range of botanical, ecological and geological questions, but recent work has revealed the importance of pollen viability in invasion biology as well. Here, we report an efficient visual method for assessing the viability of pollen using digital holographic microscopy (DHM). Imaging data reveal that quantitative phase information provided by the technique can be correlated with viability as indicated by the outcome of the colorimetric test. We successfully test this method on pollen grains of Lantana camara, a well-known alien invasive plant in the tropical world. Our results show that pollen viability may be assessed accurately without the usual staining procedure and suggest potential applications of the DHM methodology to a number of emerging areas in plant science.

The formation of a bright-field microscopic image of a transparent phase object is described in terms of elementary geometrical optics. Our approach is based on the premise that the image replicates the intensity distribution (real or virtual) at the front focal plane of the objective. The task is therefore reduced to finding the change in intensity at the focal plane caused by the object. This can be done by ray tracing complemented with the requirement of energy conservation. Despite major simplifications involved in such an analysis, it reproduces some results from the paraxial wave theory. In addition, our analysis suggests two ways of extracting quantitative phase information from bright-field images: by vertically shifting the focal plane (the approach used in the transport-of-intensity analysis) or by varying the angle of illumination. In principle, information thus obtained should allow reconstruction of the object morphology.