Background

Cryo-SEM is an irreplaceable approach for investigating biological samples in a hydrated state [1–5], and it has been proven to be a powerful tool for studying the microstructure and surface morphology of algae and cells [6–11]. There are four main procedures for sample treatment in cryo-SEM, including cryo-fixation, freeze-fracture, freeze-etching, and sputter coating [12,13]. Cryo-fixation is a technique for rapidly freezing a sample to preserve its structure in a near-native state [14,15]. Typically, a liquid nitrogen bath at atmospheric pressure, liquid nitrogen slush, liquid nitrogen under high pressure (for high-pressure freezing), and liquid ethane and propane are employed as cryogenic substances [14–17]. Freeze-fracture is a technique that fractures the cryo-fixed sample in order to reveal its internal structure, typically by a cold knife or a cryo-ultramicrotome [18]. Freeze-etching is the process of getting the fractured surface etched by sublimation of ice, which reveals fine details of the sample's structure. Sputter coating in cryo-state is similar to the conventional sputter coating, except that the sample is maintained in a cryogenic state.

There are some valuable protocols that have been published regarding plant and animal tissues as well as other biomaterials [1–6,9,13]. Nevertheless, only a limited number of protocols are available for cryo-SEM of algae. Research articles regarding cryo-SEM investigation of algae samples usually include a brief description of the cryo-SEM experiments but do not provide much information about the detailed and visualized experimental procedures. Particularly, information such as how the sample is prepared and how it is freeze-fractured is crucial for obtaining fine details of the internal structures.

The technique of freeze-fracture has been widely used in a broad range of samples, such as fluid samples, hydrated polymers and microgels, and plant tissues [18–20]. In the freeze-fracture procedure, a cold knife in a cryogenic state is commonly employed as the cutting tool: a blade with a sharp edge that is positioned above the sample. It sweeps across the top of the sample in parallel with the sample holder. When it hits the sample, preferably a prominent part or a tip, the sample is then freeze-fractured [1]. During this process, a certain type of sample holder or a specific way to hold the sample is employed. Due to the diverse characteristics of hydrated samples, such as water content, viscosity, strength, shape, or amount, various ways for sample holding are required. Consequently, more and more sample holders or approaches for holding samples are being developed. For example, Mo et al. developed a sample stage (holder) for the freeze-fracture of liquid, semi-liquid, and viscous samples, including chlorella [21]. Nonetheless, reliable protocols with easy and flexible sample-holding approaches that are compatible with various types of samples are still needed.

In this protocol, we propose an easy and reliable approach to hold and support the sample by employing a piece of filter paper as the substrate. The advantage of filter paper lies in its ability to support a thin film of sample, enabling a cold knife to make a cut and produce a clean freeze-fractured surface. This method is versatile and applicable to a broad range of samples, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto a filter paper. Along with detailed, step-by-step operations for cryo-SEM tests, the authors believe that this protocol will be helpful for researchers to commence the design and operation of their cryo-SEM experiments.