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

This study presents a robust methodology for the three-dimensional (3D) volumetric reconstruction and morphological characterization of native Chilean pollen grains using a lens-free Digital In-line Holographic Microscopy (DLHM) system. Utilizing a 532 nm laser point-source configuration and a 3.45 $\mu$m pixel pitch CMOS sensor , we achieved a geometric magnification of 50x, resulting in an effective lateral resolution of approximately 69 nm at the object plane. The complex wavefronts of $\mathit{\text{Anthemis}}\mathit{\text{cotula}}$ (chamomile), $\mathit{\text{Gevuina}}\mathit{\text{avellana}}$ (hazel), and $\mathit{\text{Conium}}\mathit{\text{maculatum}}$ (hemlock) were numerically reconstructed via the Kirchhoff-Helmholtz transform to generate high-fidelity 3D refractive index maps. Biophysical parameters were extracted with nanometric precision, with volumes ranging from $3780.2\pm 18$ $\mu$m${}^3$ to $4320.5\pm 15$ $\mu$m${}^3$. Morphological quantification identified $\mathit{\text{A.}}\mathit{\text{cotula}}$ as the least spherical species ($\mathrm{\Psi }=0.76\pm 0.03$) due to its characteristic echinate (spiny) exine, while $\mathit{\text{G.}}\mathit{\text{avellana}}$ exhibited the highest sphericity index of $0.89\pm 0.02$. These results demonstrate that the label-free retrieval of “digital fingerprints” provides a scalable alternative for automated melissopalynology and viability assessment, filling critical geographic data gaps in South American biodiversity hotspots.