Light sheet microscopy and live imaging of plants

Abstract

SummaryLight sheet fluorescence microscopy (LSFM) is increasingly used to investigate biological processes in animals as well as in plants. LSFM achieves optical sectioning by the selective illumination of a single plane of the sample with a sheet of laser light while simultaneously recording emitted fluorescence orthogonally to the illumination plane. A 3D image of the sample can then be generated with a temporal resolution ranging from seconds to several days, and at scales ranging from subcellular to whole organ. By design, LSFM provides fast imaging, and low phototoxicity, two key criteria for live imaging under physiological conditions. Despite its potential, LSFM remains underutilized in plant biology. This review aims to highlight challenges of live imaging in plants, to describe key steps in using LSFM on live plant samples and finally at providing an overview of published examples of applications of LSFM in plants.

Probing the 3D architecture of the plant nucleus with microscopy approaches: challenges and solutions

“…using quantitative nuclear reporters) over time within millimetres of depth. Light sheet microscopy has been adopted in the plant science field in recent years [70] to offer another attractive option with high temporal resolution, although this is currently at the expense of spatial resolution and is limited by computationally expensive image processing.…”

“…Note that in some cases, imaging roots at the horizontal position can be an obstacle, particularly if the study is focused on root growth or gravistimulation. To solve this issue, von Wangenhein and colleagues developed a confocal microscope setup for vertical sample mounting [69,70] .…”

“…For longterm imaging (>6 h), the setup has to implement light-, temperature-and mounting medium-control (preventing evaporation and oxidation over time) compatible with the physiological or developmental process being studied. Several suitable customized setups and controlled growth/imaging chambers have been described [69,70,151,[153][154][155]156] . So far, reports on long-term DLIm in plants are cellrather than nuclei-oriented, and imaging nuclei at high resolution will require adaptation of these methods.…”

“…Long-term nuclei imaging brings the additional challenge of tracking live nuclei that can move outside the field of view and out of focus during the experiment. Advances in the imaging of growing tissues, such as plant roots, have resulted in the development of different image processing-tracking tools that can correct the positioning of the microscope stage on the fly to maintain the sample in the field-of-view over several hours or days [69,70,158]. Recent tools customized to follow nuclear architecture dynamics in Arabidopsis roots are discussed below.…”

“…Tracking methods for root tips have been published [69,70,159,160] . They function on the same broad principle of comparing successive time-points to predict the next position and re-adjusting a motorized microscope stage accordingly.…”