Super-resolution imaging of Douglas fir xylem cell wall nanostructure using SRRF microscopy

Abstract

Background The nanostructure of plant cell walls is of significant biological and technological interest, but methods suited to imaging cell walls at the nanoscale while maintaining the natural water-saturated state are limited. Light microscopy allows imaging of wet cell walls but with spatial resolution limited to the micro-scale. Most super-resolution techniques require expensive hardware and/or special stains so are less applicable to some applications such as autofluorescence imaging of plant tissues. Results A protocol was developed for super-resolution imaging of xylem cell walls using super-resolution radial fluctuations (SRRF) microscopy combined with confocal fluorescence imaging (CLSM). We compared lignin autofluorescence imaging with acriflavin or rhodamine B staining. The SRRF technique allows imaging of wet or dry tissue with moderate improvement in resolution for autofluorescence and acriflavin staining, and a large improvement for rhodamine B staining, achieving sub 100 nm resolution based on comparison with measurements from electron microscopy. Rhodamine B staining, which represents a convolution of lignin staining and cell wall accessibility, provided remarkable new details of cell wall structural features including both circumferential and radial lamellae demonstrating nanoscale variations in lignification and cell wall porosity within secondary cell walls. Conclusions SRRF microscopy can be combined with confocal fluorescence microscopy to provide nanoscale imaging of plant cell walls using conventional stains or autofluorescence in either the wet or dry state.

Obtaining super-resolved images at the mesoscale through Super-Resolution Radial Fluctuations

“…Other work utilising SRRF often uses higher magnification values to obtain super-resolved images 21,28,29 . Increasing the magnification also increases the size of the image being processed, as each original pixel is split into a grid of smaller pixels, increasing the data contained within one image.…”

“…SRRF has been widely used for a variety of biological applications, with studies reporting an increase in spatial resolution from 235 nm to less than 100 nm in the cell wall of xylem from Douglas Fir trees, although over a limited field with only a few cells analysed per image 21 . This approximate 2-fold improvement in resolution is consistent across other work using SRRF, including the quantification and localisation of azurophilic granules in neutrophil leukocytes 22 , where the resolution of raw data was increased from 160 nm to better than 100 nm using SRRF but, again, only a few cells were captured in each image 22 .…”

Functional Complexity on a Cellular Scale: Why In Situ Analyses Are Indispensable for Our Understanding of Lignified Tissues

“…The combination of fluorescent lignin stains, such as rhodamine B, with super-resolution microscopy even goes beyond that, resolving differences in lignin quantity below 100 nm. 51 Of the numerous chromogenic and fluorescent lignin stains, only very few have been validated regarding their quantitative capacity and chemical specificity. The absorbance of the Wiesner test represents a reliable way to estimate the cell wall layer-specific G CHO concentration in situ.…”

“…Given the adequate sample preparation, the spatial resolution of these methods is close to the diffraction limit. The combination of fluorescent lignin stains, such as rhodamine B, with super-resolution microscopy even goes beyond that, resolving differences in lignin quantity below 100 nm . Of the numerous chromogenic and fluorescent lignin stains, only very few have been validated regarding their quantitative capacity and chemical specificity.…”

Exploring Lignification Complexity in Plant Cell Walls with Airyscan Super-resolution Microscopy and Bioorthogonal Chemistry

“…STORM was employed to observe cellulose microfibrils in a report, and to study the role of pectins in lobe formation in Arabidopsis pavement cells in another . Concerning lignin, its cell wall distribution was investigated in 2018 by STED microscopy combined with polyethylene glycol-rhodamine (PEG-R) conjugates, then in 2022 with super resolution radial fluctuation (SRRF) microscopy associated with lignin conventional stains or autofluorescence . To the best of our knowledge, the latter are the only two reported attempts at super-resolution microscopy on the topic of lignin.…”