High‐resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy

Abstract

SUMMARYMost plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.

Lateral root morphogenesis is dependent on the mechanical properties of the overlaying tissues

“…LRP 2D shapes used for statistical analysis were defined by Bezier curves using 26 control points per primordium. Light-sheet microscopy was performed as described previously (9). Images were taken with either a Carl Zeiss W N-Achroplan 20×/0.5 (Fig.…”

“…1B). To independently validate our observations, we used light sheet fluorescence (LSF) microscopy to noninvasively image these tangential divisions (9). LSF microscopy was used to monitor early stage primordia expressing the plasma membrane marker pPIN1::PIN1-GFP (15).…”

“…Recent advances in live biological imaging and image analysis (8,9) now make it possible to address how Arabidopsis LRPs are built in three and four dimensions. Here, we report how 3D/4D image reconstruction reveals important divisions that are responsible for the transition from a bilateral to a radial symmetry of the LRP.…”

Microtubule networks for plant cell division

“…Future insights into the organization and functioning of plant microtubule networks for cell division will be aided by new developments on model systems and light microscopy. In particular the continuing development of plane-illumination microscopy will allow for the long-term visualization of 3 dimensional microtubule networks within developing plant tissues (Maizel et al 2011). Cell division in higher plants occurs in complex meristematic tissues in which microscopic observations of cell division are often hindered by several layers of overlaying cells.…”

Drawing Out by Drawing Into: Representation and Partnership in a Design-Science Collaboration

“…He has worked extensively on microscopy and imaging technologies (Federici et al 2012;Maizel et al 2011), including those that Benjamin and he used in their collaboration. His considerable skill with these methods and instruments helps explain the widespread attention his images have gained both inside and outside the biological sciences (Osterrieder 2013;Parsons 2012).…”