Preparation and staining of hard seed tissue for backscatter imaging
Hard seed coat material is extremely difficult to prepare for observation at the EM level. Seed coat segments are usually removed for embedding and sectioning, which results in loss of tissue juxtaposition. Observation and monitoring reactions of the intact seed coat under experimental procedures such as tracking dye penetration into seeds make seed coat dissection unfeasible. Efforts to apply analytical EM methods such as STEM/EDS analysis, are restricted by the limiting effect of section thickness on X-ray signal. We investigated the ultrastructure of dry seeds by treating them with various metallic staining solutions and observing the trimmed block faces with the SEM in backscatter mode. Good structural information was obtained as well as visualization of a specific tissue layer that accumulates lead.Intact seeds were incubated in several aqueous solutions including: 2% w/v uranyl acetate; 1% w/v OsO4; 2% ferrous chloride (Followed by 5% potassium ferricyanide to form an insoluble precipitate) ; and various lead solutions including Reynolds lead citrate.