Preparation of plants for developmental and cellular imaging by light-sheet microscopy

Abstract

Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.

Integrating cell biology and proteomic approaches in plants

“…A great benefit of this technique is that low resolution immunofluorescent data can be combined and correlated with high resolution immunogold electron microscopy results [22]. Moreover, progress in microscopic imaging methods including high-throughput microscopic analyses, light sheet and super-resolution microscopy provided new tools for the subcellular investigation of proteins in better detail and time scale [23,24].…”

Light sheet microscopy and live imaging of plants

“…Plant samples can be grown either directly into holders, such as glass capillaries or fluorinated ethylene propylene (FEP) tubes (Maizel et al, 2011;Sena et al, 2011;Kaufmann et al, 2012;Ovečka et al, 2015), or on a solid medium and then later introduced to a holder such a glass capillary or customized holding device (Fig. 1D) (Maizel et al, 2011;Kaufmann et al, 2012;Jeandupeux et al, 2015;Ovečka et al, 2015). An example of this can be seen in the imaging of the initial steps of plant germination, which is performed by positioning seeds inside jellified medium within a glass or FEP tube (Fig.…”

Quantitative Reflection Imaging for the Morphology and Dynamics of Live Aplysia californica Pedal Ganglion Neurons Cultured on Nanostructured Plasmonic Crystals

“…23–25 Fluorescence imaging, however, can be complicated by the toxicity associated with introducing foreign fluorescent labels into the system and the susceptibility of fluorophores to photobleaching. 9, 26, 27 Phase contrast microscopy 28, 29 and differential interference contrast microscopy 30, 31 make it possible to visualize unstained biological specimens with enhanced contrast and resolution. These imaging methods provide a useful means to study cellular structures and processes, albeit with lesser quantitative capabilities for analyzing live cell morphology.…”