Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

Abstract

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial-as well as temporal resolution over prolonged periods of time, which may cause phototoxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

Global analysis of an exponential model of cell proliferation for estimation of cell cycle duration in the root apical meristem of angiosperms

“…Analysis of heterogeneity of T in sister cells within the maize RAM showed they do not vary by more than 15-20 % (Ivanov, 1971). Recent time-lapse studies in arabidopsis showed that variation in sister cell T averaged 8.3 % (von Wangenheim et al, 2017b; data extracted from their Video 3, n = 6 sister groups). Another source of heterogeneity is that some cell types (metaxylem in monocots) stop proliferating earlier, but the fraction of these cells is also not significant.…”

Advances in Imaging Plant Cell Dynamics

“…However, embedding of plant aerial parts is not compatible with proper physiological conditions, which are necessary for long-term imaging. Recently developed open systems for plant preparation and maintenance during LSFM long-term imaging von Wangenheim et al, 2017) ensure that plants are maintained in the microscope vertically oriented (according to the gravity vector). Simultaneously, continuous root growth is supported by appropriate culture medium, and the aerial part grows freely in the air.…”

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

“…While perfusion systems will remain the method of choice for wash-out experiments, the imaging chamber has been used successfully for the administration of dexamethasone 12 and other compounds. One note, while this article was in preparation von Wagenheim and colleagues 18 described a chamber for imaging lateral root development using light-sheet microscopy.…”