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.