Three-Dimensional Volumetric Reconstruction of Native Chilean Pollen via Lens-Free Digital In-line Holographic Microscopy

Abstract

This study presents a methodology for the three-dimensional volumetric reconstruction and morphological characterization of native Chilean pollen grains using lens-free Digital In-line Holographic Microscopy (DLHM). A 532 nm point source, a 3.45 $\mu$m pixel pitch CMOS sensor and a source--sample distance of $0.46$ mm with a $25$ mm stand-off give a magnification of $\simeq 55\times$ and an effective pixel of 62.9 nm. Wavefronts of \textit{Anthemis cotula}, \textit{Gevuina avellana} and \textit{Conium maculatum} were reconstructed by the Kirchhoff-Helmholtz transform, yielding volumes from $3780.2 \pm 18$ to $4320.5 \pm 15$ $\mu$m$^3$ and sphericities from $0.76 \pm 0.03$ for the echinate \textit{A. cotula} to $0.89 \pm 0.02$ for \textit{G. avellana}. Three independent targets make these reconstructions traceable. Certified polystyrene microspheres ($\varnothing = 20.0$ $\mu$m) make the source--sample distance an optically retrieved quantity, the 2.5D height map returning $2068$ $\mu$m$^3$ against an analytical $2094.4$ $\mu$m$^3$. A human erythrocyte, reconstructed with a haemoglobin refractive-index increment taken from the literature rather than fitted, returns $88.9$ fL with a $2.36$ $\mu$m rim and a $1.03$ $\mu$m dimple, within physiological range. Finally, transport-of-intensity retrieval on bright-field micrographs from a separate, independently calibrated instrument reproduces the projected morphology of \textit{G. avellana}, though no volume follows from it. These results support label-free "digital fingerprints" for automated melissopalynology in a South American biodiversity hotspot.