A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope

Abstract

A comparative investigation of techniques for the preparation of soft botanical tissue for the scanning electron microscope has been carried out using the leaves and petals of Pelargonium zonale as test specimens. Twelve different preparative procedures involving combinations of fixation, dehydration, air drying, freeze drying, critical point drying, coating methods, replicas and a temperature controlled specimen stage were tested.

Optical sectioning and 3D reconstructions as an alternative to scanning electron microscopy for analysis of cell shape

“…The literature is rich with different methods for visualizing cell structure. Parsons et al (1974) compared 12 different sample preparation methods for visualizing cells and found that with no time or equipment constraints, the best method was the use of untreated material. However, this approach requires that tissue be examined immediately after collection, which in some cases may not be feasible.…”

Critical point drying for scanning electron microscopy: a semi‐automatic method of preparing biological specimens

“…It is likely that until an inert water miscible fluid with a low Tc is obtained, CPD of most biological material will involve the use of intermediate fluids despite the evidence of some shrinkage or swelling caused by the commonly used intermediate fluids (Boyde et al, 1977;Gusnard & Kirshner, 1977). The importance of minimizing specimen damage at the fluid substitution stage has been stressed previously (Parsons et al, 1974), and the procedure adopted in the present work with Pelargonium zonale is believed to involve minimum damage (as indicated by the similar appearance in the SEM of specimens prepared by CPD, freeze-drying and when viewed on the cold stage). It is essential for investigators using CPD to select a suitable intermediate fluid/ transitional fluid system with regard to the objectives of their investigation.…”

Effects of four different processing techniques on the microstructure of potatoes: Comparison with fresh samples in the ESEM

“…Examination of most botanical tissues using conventional SEM and ultra high resolution field emission (FESEM) requires careful sample preparation in order to fully dehydrate the sample without significant tissue damage. The collapse of delicate cellular structures and the introduction of drying artifacts must be avoided (e.g., Parsons et al, 1973;OBrien and Mc-Cully, 1981; Mueller and Hermann, 1990; Goldstein et al, 1992;Haggis, 1992; Peters and Pohl, 1992). Until direct imaging of unprocessed tissues became possible using ESEM (Danilatos, 19811, botanists and biologists had relied on complex processing procedures designed to preserve cell structure.…”

“…Despite the importance of various processing methods for conventional and high resolution SEM, there are surprisingly few investigations which directly compare and contrast several different processing techniques on one sample. Notable exceptions are the studies of Parsons et al (1973) and Hardy et al (1992) who examined flower petals and grass leaves, respectively. In addition, whilst the improvements in imaging wet, hydrated biological samples using ESEM have been reported by many authors (e.g., Wallace et al, 1992;Klose et al, 1992) only a few compare unprocessed ESEM samples with samples prepared using conventional techniques (Danilatos, 1981;O'Brien et al, 1992).…”