An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology

Abstract

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.

Advances in Imaging Plant Cell Dynamics

“…This may hamper the visualization of the subcellular architecture of plant samples not adequately coated by the observation medium. It can be avoided by mounting plants in an oxygen-saturated perfluorocarbon (Littlejohn et al, 2010(Littlejohn et al, , 2014Littlejohn and Love, 2012;Chi and Ambrose, 2016). SIM also is prone to optical artifacts that may be caused by inaccurate grid movements (Langhorst et al, 2009), and resolving capacity depends on the labeling quality and the signal-to-noise ratio (SNR) of the sample.…”

Making microscopy count: quantitative light microscopy of dynamic processes in living plants

“…() and Littlejohn et al . (). To maximise IR transmission, the standard galvanometer scanning mirrors were replaced with silver galvanometric mirrors, the tube lens was replaced with a MgF 2 coated lens and the confocal dichroic was replaced by a silver mirror with high reflectivity throughout the visible and NIR (21010, Chroma Technologies, USA).…”

In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy

“…Fresh, excised plant leaves were mounted in perfluorodecalin (Littlejohn et al, 2010) prior to imaging, which has been shown not to interfere with Raman-based imaging in plants (Mansfield et al, 2013;Littlejohn et al, 2014).…”