Microscopic 3D plant imaging with high-resolution optical coherence tomography
J. de Wit (TU Delft - ImPhys/Kalkman group, TU Delft - ImPhys/Computational Imaging)
J. Kalkman – Promotor (TU Delft - ImPhys/Computational Imaging, TU Delft - ImPhys/Kalkman group)
S. Stallinga – Promotor (TU Delft - ImPhys/Imaging Physics)
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Abstract
This thesis explores the application of optical coherence tomography (OCT) for high-resolution, in-vivo 3D imaging of plants and plant pathogens. OCT offers label-free optical sectioning and complements conventional microscopy, particularly for visualizing unlabeled pathogens within plant tissues. However, imaging small pathogens requires improvements in depth, resolution, and specificity, which form the central focus of this work.
Plant leaves contain air-filled cavities for gas exchange, which scatter light and distort the wavefront, limiting imaging depth. In Chapter 2, we show that infiltrating leaves with water or perfluorodecalin significantly reduces these effects, extending OCT imaging depth to the full leaf cross-section of several hundred micrometers and enabling quantitative measurement of leaf thickness.
OCT depth information is typically obtained via Fourier-transform-based spectral-domain processing, limiting axial resolution to the coherence length of the source. In Chapter 3, we optimize a spectral estimation (SE) method, the iterative adaptive approach (IAA), which improves axial resolution by a factor of 2–10, depending on signal-to-noise ratio. IAA preserves intensity and speckle statistics and allows sub-second B-scan reconstruction.
Chapter 4 extends SE-OCT with coherent refocusing and computational aberration correction, achieving a lateral resolution of 0.8 μm and improving axial resolution from 8 μm to 1.5 μm. Depth-of-field extension by a factor of 20 enables high-resolution 3D imaging across a large volume.
While conventional OCT images tissue morphology based on scattering, distinguishing pathogens from host tissue remains challenging. Chapter 5 introduces dynamic OCT (dOCT), which uses temporal speckle fluctuations to generate functional contrast. Bremia lactucae, a downy mildew pathogen in lettuce, exhibits intermediate-frequency speckle fluctuations (0.7–5.5 Hz), while plant tissue remains largely static. This contrast allows imaging and segmentation of pathogen structures, quantifying infection levels and revealing differences in resistance among lettuce genotypes. The in-vivo capability of OCT is demonstrated by tracking infection progression and individual hyphal growth over several days.
The concluding chapter outlines future directions, including phase-leakage reduction in phase-sensitive OCT and 2D SE-OCT for lateral resolution enhancement. Overall, this thesis demonstrates that OCT, enhanced through optical clearing, spectral estimation, and dynamic contrast, provides biologically relevant, high-resolution 3D imaging of plants and pathogens. These methods offer quantitative insights into plant-pathogen interactions and lay the groundwork for further functional imaging in plant biology.