Cross-scale Imaging of Live Botanical Specimens using Fourier Ptychographic Microscopy

Abstract

Light microscopic imaging of botanical specimens is typically performed using diffraction-limited methods which can image either a small field of view (FOV) with a shallow depth of field (DOF) at high resolution, or a large FOV and large DOF at low resolution. As such, many of the important small features, such as cells in the root cap of Arabidopsis, chloroplasts in algae and leaf guard cells, cannot be resolved in high numbers for meaningful statistical interpretation of results. Fourier ptychographic microscopy (FPM) is an accessible low-cost solution for large format bioimaging which makes use of multiangle illumination and iterative phase retrieval to recover high-resolution estimates of sample phase and amplitude. To date much FPM research has focused on optimising hardware and image reconstruction techniques with biological and biomedical applications limited primarily to mammalian cell cultures and histological tissue sections. We have, for the first time, applied FPM for label-free imaging of these large, living botanical specimens to investigate cellular and sub-cellular structures over multiple length scales simultaneously, with the aim of resolving structures that cannot be seen with diffraction-limited methods over a large field of view. With our FPM system we demonstrate imaging of living samples mounted in water with a resolution of 615 nm over a 3.6 mm diagonal FOV, producing a 9-fold improvement in resolution compared to conventional brightfield imaging. This allows for individual root cap cells of Arabidopsis thaliana to be resolved within the context or larger root structures, individual chloroplasts identified in large section of Spirogyra varians, and high-resolution colour imaging of Tradescantia zebrina stomata and chloroplasts across large leaf sections.