In situ ESEM using 3-D printed and adapted accessories to observe living plantlets and their interaction with enzyme and fungus

[Display omitted] •The specimen holder of an ESEM could be tailored to accommodate the living plantlets in the microscope chamber and perform in situ observations.•Optimization of ESEM imaging parameters ensures the viability of seedlings during repetitive observation and the time-course study of their growth and development.•The ESEM reveals the effect of exogenously applied polygalacturonase on the living Arabidopsis epidermal cells.•The interaction of fungus Verticillium dahliae with live flax plantlets on the surface is visualized using the in situ ESEM. This paper describes an innovative way of using environmental scanning electron microscopy (ESEM) and the development of a suitable accessory to perform in situ observation of living seedlings in the ESEM. We provide details on fabrication of an accessory that proved to be essential for such experiments but inexpensive and easy to build in the laboratory, and present our in situ observations of the tissue and cell surfaces. Sample-specific configurations and optimized tuning of the ESEM were defined to maintain Arabidopsis and flax seedlings viable throughout repetitive exposure to the imaging conditions in the microscope chamber. This method permitted us to identify cells and tissues of the live plantlets and characterize their surface morphology during their early stage of growth and development. We could extend the application of this technique, to visualize the response of living cells and tissues to exogenous enzymatic treatments with polygalacturonase in Arabidopsis, and their interaction with hyphae of the wilt fungus Verticillium dahliae during artificial infection in flax plantlets. Our results provide an incentive to the use of the ESEM for in situ studies in plant science and a guide for researchers to optimize their electron microscopy observation in the relevant fields.