Using a vibrating microtome to prepare Mentha x piperita shoot tip sections for live-tissue microscopy experiments

Authors: KRECKEL, HEIDI - Colorado State University; OCHOA-CASTILLO, ALBERT - Colorado State University; VILLANUEVA, STEPHANIE - Colorado State University; Volk, Gayle; LEVINGER, NANCY - Colorado State University

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2025
Publication Date: 4/29/2025
Citation: Kreckel, H.D., Ochoa-Castillo, A., Villanueva, S., Volk, G.M., Levinger, N.E. 2025. Using a vibrating microtome to prepare Mentha x piperita shoot tip sections for live-tissue microscopy experiments. Plant Cell Tissue and Organ Culture. 161. Article e40. https://doi.org/10.1007/s11240-025-03058-7.
DOI: https://doi.org/10.1007/s11240-025-03058-7

Interpretive Summary: Plant cell imaging techniques have been used to understand the movement of solutes within and between cells. These methods allow for the visualization of the uptake of cryoprotectant solution components into the meristems and surrounding immature leaves of living plant shoot tips. This is challenging because shoot tips have many densely packed cells that create an opaque tissue. Previously, experiments were performed using shoot tips that were fixed, embedded into resin, sectioned, and then stained. This manuscript describes the use of a vibrating microtome to section living shoot tips, allowing real-time cellular dynamics to be observed. The vibrating microtome produces sections that are more uniform and reproducible than those excised by hand. Both brightfield and fluorescence microscopy techniques were used to confirm sample viability and to demonstrate the cellular anatomy that can be observed.

Technical Abstract: Live plant tissue imaging can be challenging due to thick, opaque tissue and interference from endogenous fluorescence. Many studies probe fixed samples, which kills the tissue and precludes observation of real time dynamics. Preparation of plant shoot tips, which preserves the genetics of a parent plant, for microscopy experiments presents additional and unique challenges: (1) producing shoot tips of sufficiently high quality by hand excision requires committed practice to obtain sufficient expertise, (2) shoot tip thickness and size vary making subcellular analysis difficult, and (3) desired sample orientation is unreliable and difficult to ensure. In this work, we demonstrate the preparation of living peppermint shoot tip sections for microscopy experiments using a vibrating microtome, which dramatically reduces training time and yields significantly better sample reproducibility. We use a combination of brightfield and fluorescence microscopies to confirm sample viability and demonstrate successful histological staining without the use of fixatives or toxic additives to increase the permeation by large, hydrophobic stains or dyes.