1. Introduction

To obtain a high resolution SEM image of small objects, such as bacteria and eukaryotic cells, proper preparation of a sample is essential. Since it is crucial to maintain a vacuum of at least 10 −4 Pa and to prevent contamination, biological samples must be carefully processed and dehydrated. An inadequate preparation of a specimen may result in shrinkage and deformation of cells, leading to incorrect results and misleading interpretations ^[1]. The process of preparing biological samples for SEM imaging consists of several stages, and the main ones include fixation, dehydration and drying. In addition, specimens are typically sputter-coated with a conducting film, since the investigated surface should be conductive to prevent image distortion caused by an electron charging effect ^[2].

An alternative to conventional SEM imaging performed under reduced pressure is the use of an environmental scanning electron microscopy (ESEM), in which the pressure around the sample is increased to 10–20 torr. In this method, gas particles in the chamber are ionised facilitating a free flow of current, which in turn allows for imaging of wet and non-conductive samples. The obvious advantage of imaging biological materials with ESEM is the ability to collect an image with minimal sample preparation ^[3][4]. However, a negative effect of humidity is still observed at higher pressures, and the presence of condensed water layer on a sample can result in low contrast and poor visibility of fine details of cells. Moreover, applied conditions are at a burden for biological materials, therefore it is generally accepted that a single ESEM sample can be only viewed once ^[5]. According to these limitations, the major challenge associated with ESEM is to dry the samples while preventing structural damage to investigated materials. Common drying methods include critical point drying, freeze-drying and direct air-drying after dehydration with alcohol.

2. Specifics

In a freeze-drying method, a specimen in the aqueous phase is quickly frozen and transferred to a special chamber where the temperature is kept below −80 °C. The frozen substance sublimates to the gas phase and then is absorbed or removed by vacuum. The process of freeze-drying can take from several hours to several days depending on sample size, temperature, and pressure. The major advantage of this method is limited shrinkage, especially when compared with CPD. However, some disadvantages include the need for a special equipment, problems with rapid freezing and transfer of the sample, long time needed to dry biological samples, and the presence of a sediment remaining on the sample surfaces. Additionally, freeze-drying can cause distortions and damage due to the formation of ice crystals ^[1][6][7][8].

The above-mentioned drying techniques, although giving excellent results for certain biological samples, are not always suitable for examining every microorganism or tissue. Moreover, they require complex, specialised, and expensive equipment. Therefore, the easiest and most effective way for drying of biological samples is a simple air-drying ^[9][10]. Although air-drying carries the risk of an excessive shrinkage, cracking, and collapse of fragile structures such as cilia and flagella, the use of solvents such as ethanol or HMDS in the dehydration phase enables to apply air-drying without damaging tested materials ^[8].