Background

Scanning electron microscopy (SEM) refers to the use of an electron beam apparatus and a pattern inspection apparatus to image a sample surface (Yan, 2010). The first SEM came on the market in 1965 when the Cambridge Instrument Company launched a commercial instrument (McMullan, 1995). Specifically, biological samples must be dehydrated before entering the SEM chamber (Echlin, 1971), otherwise water vapor contaminates the electron microscope vacuum system. Before cryopreservation was invented, freeze-drying and critical point drying were two commonly used methods (Sargent, 1986). These pretreatments were inevitably associated with sample distortion, shrinkage, or loss of inner cellular soluble components (Reference 1; Echlin, 1971); additionally, they were time-consuming and laborious.

The water in a biological material generally forms ice crystals when the sample freezes at 0°C or below; however, the water forms a glasslike structure when the material is cooled at a very high cooling rate (Rey, 1960; Binder, 2014; Limmer and Chandler, 2014). In comparison with the crystal structure, this glasslike, amorphous water minimizes damage to the cell structure and mechanical properties (Vega-Gálvez et al., 2008); therefore, cryo-scanning electron microscopy (cryo-SEM) was devised to exploit this advantage.

An exceptional paper presented a detailed and comprehensive description of cryo-SEM development and its application in biology (Read and Jeffree, 1991). In short, cryo-SEM was first performed in 1960 but was not widely known until 1970 (Echlin, 1971). Commercial cryopreparation systems were available for SEMs in the 1980s; henceforth, cryo-SEM technology became routine and was commonly used in biological research (Read and Jeffree, 1991). Additionally, cryo-SEM was widely used in the field of botany for observations of the surface or freeze-etched fractions of roots (Vartanian et al., 1983; Webb and Jackson, 1986; Ryan et al.,1998; Dolan et al., 1994; Foreman and Dolan, 2001; Müller and Schmidt, 2004; Ding et al., 2009; Yi et al., 2010; Zhiming et al., 2011; Huang et al., 2013; Zou et al., 2015; Giri et al., 2018; Wang et al., 2019; Zenone et al., 2020), stems (Echlin, 1971), vessels (Utsumi et al., 1998), leaves (Sargent, 1983), shoot apexes (Kaneko, 1985), glandular trichomes (Kaneko, 1985), stamen hair cells (Kaneko, 1985), stigmas (Kaneko, 1985), petals (Wang et al., 2019), pollen grains (Echlin, 1971; Berger et al., 1998), stomatal pores (Echlin, 1971), berry skins (Brizzolara et al., 2020), seeds (Yu et al., 2014), etc. Moreover, cryo-SEM has been applied to the visualization of root-fungal interactions (Refshauge et al., 2006).

Root hairs are a type of tubular protuberant cell that diverges from specific root epidermal cells (Ishida et al., 2008; Kim and Dolan, 2016). They are capable of increasing root and soil contact areas and improving the efficiency of water and nutrient absorption while providing a place for the plant to interact with soil microorganisms (Larkin et al., 2003).

Without cryo-SEM, it is difficult to obtain favorable results for root hairs because of the large proportion of water inside root hairs. Root hair shape maintenance is dependent on turgor pressure driven by inner water (Mendrinna and Persson, 2015). Essentially, some samples, such as primary roots (Lefebvre, 1985), leaves (Eveling and McCall, 1983; Sargent, 1983), petals (Chen and Meyerowitz, 1999), and stamens (Chen and Meyerowitz, 1999), can remain intact after drying treatment and be photographed by SEM.

Nevertheless, with effective cryo-SEM technology, root hairs inevitably shrink and/or bend during cryo-preparation (Dolan et al., 1994; Czarnota et al., 2003; Cocozza et al., 2008; Zou et al., 2015); therefore, the process of preparing cryo-SEM samples is extremely important. In the following sections, some operations are highlighted and introduced in detail. In the foreseeable future, this technology will be suitable for stamen filament samples and other samples similar to root hair.

Advantages of Cryo-SEM:

1. It is suitable for tissue samples with a high moisture content.

2. Samples do not need to be fixed or dehydrated in advance.

3. It is proficient at revealing details that optical microscopy cannot.

4. Detailed quantitative parameters of the root hairs can be obtained.

5. The interior cell structure can be studied.
   

1. Rice roots must be cultured in an agar plate.

2. Root sample throughput is fairly low as compared with other methods.

3. Sample pretreatment requires professional operation skills.

4. Price of sample pretreatment is relatively high.