Historical background of microscopy:

Owing to the low resolving power of the naked human eye, it had been understood long back that visualisation of important microscopic features required the aid of some special “equipment”. The development of the first primitive compound microscope dates back to the seventeenth century when a Dutch spectacle maker observed that placing a pair of lenses in a tube makes objects at the other end appear bigger. Later in 1676, Antony van Leeuwenhoek carried out a couple of improvements in the already existing microscope, conferring it a much higher magnification. While observing a piece of cloth with the newly designed microscope, Leeuwenhoek miraculously made the ground-breaking discovery of “bacteria”, thereby creating the vastest realm of Biology, “Microbiology” (Poppick, 2017). This primitive model of the light microscope must have had a wide range of limitations, but the basic principle of light microscopy has remained the same for centuries now.

With excessive degrees of development in the field of imaging, highly sophisticated models of microscope have been designed in the past few decades, enhancing the level of research in life sciences by multiple folds. The electron microscope was first invented in 1931 by Max Knoll and Ernst Ruska at the Berlin Technical Institute. Utilising an electron beam to generate the image of a specimen instead of “light beams” (optical microscopy) helped in achieving resolutions as low as 10nm, which was further reduced to 2nm in 1944. This innovation overcame the challenge of high edge resolution that could not be achieved earlier due to limitations of visible light. The ultimate aim of obtaining atomic resolution was incrementally achieved over the course of development, paving way for extremely high quality of sample imaging and analysis (Palucka, 2002)

There are two broad categories of electron microscopes, the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM), which were sequentially developed around the same time frame. While the latter provides the viewer with highly magnified image of the external surface of an organism or object, the TEM is extensively used to study internal structures. The TEM is considered to be analogous to the conventional light microscope, in which electron beams assist in imaging thin cross-sections of specimen. Along with several practical advantages, the TEM is mostly used to image cell structures, organisation of protein molecules, and viral and bacterial cytoskeleton. The SEM took a longer course before being commercially available in 1965, and since then it has been the most widely used form of EM in the field of Biology, specifically due to its ability to confer complete structural image of a microorganism, cell, or any other object of study (Mohammed and Abdullah, 2019).

Scanning Electron Microscope (SEM):

Structurally, the SEM comprises of an electron gun for the emission of high-energy electrons, a column for electrons to travel through the electromagnetic lenses, deflection coils necessary for scanning, a backscatter electron detector for secondary electrons, a sample chamber, and a well-networked computer system to control the electron beam and to visualize the image on a screen (Fig 1).

SEM analysis is initiated with the application of high voltage electron beam through the electron gun to produce a focused spot size (usually < 10 nm) on the specimen to be studied. Movement of the scanning coils directs the electronbeam linearly to form a rectangular region within the specimen, the area of the region being dependent on the required magnification (higher magnification, smaller area). In modern SEMs, the working distance, that is the distance between the last lens and the surface of the specimen, is usually automated. The electron detector is used to collect both backscattered electrons (BSE) and secondary electrons (SE) in case of a positive voltage, and only BSE in case of anegative voltage application. The SEs are usually low energy electrons while BSEs carry high energy which helps in obtaining better contrast. The signals, displayed on the screen, can be modulated by the operator to obtain a clear image. A magnification of around 10000x is usually applied if smaller details are to be observed. Low accelerating voltages (5kV) result in images with more of surface details whereas, accelerating voltages in the range of 15-30kV lead to deeper penetration of the electrons into the specimen and give details of the interior (Mohammed and Abdullah, 2019). The resolution of the image is usually dependent on the quality of the EM sample, technical parameters of the microscope, and motive of study.

Broadly speaking, the workflow associated with conventional SEM technique can be depicted as follows (CCI, 2020):

In contrast to TEM in which samples have to be sectioned into extremely thin layers/profiles to allow electrons to penetrate through, SEM samples are usually not cut into cross-sections since this technique is more focused on imaging the external structure.

Imaging of microbial cells for identification of novel pathogens or viral diagnostics is chiefly based on electron microscopy in modern times. Although conventional methods utilised negative staining in TEM to visualise microbialcultures, a major disadvantage of this protocol was that very high concentrations of bacterial/viral cells were necessaryfor generation of clear images. In the past, problems with using SEM for microbial identification included difficulties in sample preparation. However, with the recent advent of high-quality polycarbonate filters (consisting of nanopores), microbial specimens can be conveniently collected on these filters and their surfaces can be accurately imaged through SEM. The requirement of a conducting surface for acquiring magnifications greater than 1000X and complete dehydration of biological samples, posed two of the biggest hurdles for obtaining high-performance SEM images (Golding et al. 2016). Sample drying by flash freezing or high-pressure freezing, often causes problems leading todisorientation or shrinkage of the sample. Such conventional wet sample processing also leads to mechanical disruption and loss of highly diffusible cellular elements. Therefore, alternative techniques of using wet samples are incorporated to study environmental specimens and are collectively termed as Environmental SEM (ESEM). This protocol of “wet-SEM” does not allow magnification beyond 1000x and is therefore not apt for observing specialised features such as flagella on microbial surfaces. (Golding et al. 2016).

Although high pressure freezing and ESEM techniques have enabled clear imaging of majority of wet biological samples(specimens with high water content), the most widely practiced mode of sample preparation involves cryogenic treatment and the combined procedure is known as cryo-SEM. In addition to wet specimens, the cryo-technique serves to obtain images of beamsensitive samples. The specimen to be studied is rapidly cooled and shifted to the cold stage within the preparation chamber under strictly vacuum conditions. Following this, it is mounted onto the SEM chamber and sublimation of the sample is carried out to reveal minute details. Finally, the specimen is made to undergo conductive metal coating by sputtering. Throughout the process of cryo-sample preparation, a constant temperature of -140℃ is maintained (Technical Data Sheets, EMS Catalogue number: PP3010, Cryo-SEM Preparation System, 2020).

An established research reported by Golding et al. in 2016 described a method of using a diluted ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate, instead of sputter coating. The high vacuum conditions of an SEM keep these ionic liquids in a conductive liquid state. They further demonstrated that these liquids confer a contrast to the image which is comparable to the ones generated by metal-coated specimens. However, for optimum results, a prior coating of the sample with polycarbonate filters, was observed to be essential. The phenomenal benefit of this option of usingionic liquids is that it can be carried out in biological safety cabinets and is a much safer alternative to the metal coatingof infectious bacteria and viruses. This is majorly because sputter coating of virulent pathogens in vacuum conditions creates a high possibility for the generation of infectious aerosols. This method is also rapid and reproducible (Golding et al., 2016).

SEM in Life Sciences

Scanning electron microscopy is universally used in the field of biology today. Owing to the ease of modulating image resolution, highly accurate morphological and function analyses of the specimen have now been made possible through SEM. A few of the mention-worthy examples of life science-related research, where this type of microscopy has majorly contributed, can be summarised as follows:

1. Structural Biology:

In a work carried out by Wu et al. in 2014, they have studied Staphylococcus aureus biofilms through cryo-SEM. Due to very high-water content of biofilms, such samples have to be cryo-fixed. Four samples of S. aureus biofilms were made to undergo four different treatments involving air-drying, freezing with liquid nitrogen, high-pressure freezing with liquid nitrogen, and plunging in ethane. The resulting 3-D images from samples frozen with liquid nitrogen under high pressure, helped in understanding the organisation of extracellular matrix (ECM) of the biofilm and provided a visual evidence of the distribution of S. aureus colonies in the various layers of the biofilm. This study was exemplary in establishing theimportance of cryo-SEM and highpressure freezing of sample for high-quality visualisation, and moreover re-stated the role of electron microscopy in structural biology.

2. Diagnostics:

The SEM also plays an indispensable part in the study of bone microarchitecture. Backscattering electrons (BSE) mode of the SEM allows distinct imaging of mineralised and unmineralized components of the bone structure. Age-related diseases which are associated with demineralisation of bones, such as osteopetrosis or osteomalacia, can be easily identified by BSE-SEM. SEM also aids in the diagnosis of improper bone formation in younger individuals. SE-SEM (scattered electrons) is well suited for morphological analysis of bone structure, including collagen organisation, bone metabolic activity, and local bone morphology analysis (Shah et al., 2019).

3. Functional and morphological studies:

In another significant work by Bergen et al., SEM helped in analysing the role of the Golgi matrix protein, Giantin, in regulating cilia functions in Zebrafish. The samples were pre-fixed with 4% paraformaldehyde, dehydrated with ethanol, and then critical point drying was carried out to remove the extra ethanol. As a result of a set of experiments performed by them along with the image of olfactory pit neuroepithelial cilia obtained through SEM helped them in concluding the definite contribution of Giantin in the process and maintenance ofciliogenesis. Several other extremely crucial research in the field of Biology, have been established based on images produced by SEM.

Over the past two decades, several extremely sophisticated modifications have been introduced in the SEM to allow high-accuracy 3-Dimensional analysis of biological specimens. The first modified model, serial block-face imaging SEM (SBF-SEM), was developed by Winfried Denk. This microscope uses an automated ultramicrotome located in the SEM chamber for removing thin sections from its block surface. It utilises a very high-resolution detector for capturing 3-D images at magnifications >32000x, with the X- and Y- axes resolutions lower than the Z-axes profiles. This model of SEM is widely used for analysis of brain samples in neuroscience-related research. The second improvised model is known as the focused ion or plasma beam (FIB), in which high-energy Gallium ions are used for milling sections of hard samples on the block surface of the SEM chamber. Subsequent capturing of these sections allows precise 3-D constructions of high resolutions, which helps in better understanding of essential biological questions (Bushby et al.,2011). The primary difference between these two models lies in the technique of sectioning and resolutions of the Z-axes. The ultimate goal of congregating live sample light microscopy with 3D-SEM to achieve time specific ultrastructural details of biological samples, was first trialled by Godman et al. in 1960. The contemporary developments in 3D-SEM (discussed previously) opened up new realms in the field of correlative light and electron microscopy (CLEM). New advances in the spectrum of CLEM would definitely contribute a great deal towards carving out in-depth studies related to structural and functional Biology (Kremer et al., 2014).

References:

Bushby A.J., Ping K.M., Young R.D., Pinali C., Knupp C., and Quantock A.J. (2011) Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat Protoc. Vol. 6. 845-58. http://doi.org/10.1038/nprot.2011.332.

Golding C., Lamboo L., Beniac D., and Booth T. (2016). The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci Rep. Vol 6, 26516. https://doi.org/10.1038/srep26516

Kremer A., Lippens S., Bartunkova S., Asselbergh B., BlanpaiN C., Fendrych M., Goossens A., Holt M., Janssens S., Krols M., Larsimont J.C., Guire Mc., Nowack M.K., Saelens X., Schertel A., Schepens B., Slezak M., Timmerman V., Theunis C., Vanbrempt R., Visser Y., and Gu C.J. (2014). Developing 3D-SEM in a broadbiological context (2014). Journal of Microscopy. Vol. 259. 80–96. http://doi.org/10.1111/jmi.12211

Mohammed A., and Abdullah A. (2019) Scanning Electron Microscopy (SEM): A Review. In: Proceedings of 2018 international conference on hydraulics and pneumatics–Hervex.

Shah F., Ruscsák K., and Palmquist A. (2019) 50 years of scanning electron microscopy of bone—a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy. Bone Res. Vol 7, 15. https://doi.org/10.1038/s41413-019-0053-z

Website:

https://cf.gu.se/english/centre_for_cellular_imaging/electron- microscopy/samplepreparation/specimen-preparation-for-scanning-electron- microscopy--sem (Manual for sample preparation of EM, accessed in February, 2021)

ACKNOWLEDGEMENT: Special thanks to Nikon Imaging Centre, King’s College London, London, UK and to Prof. Stephen Sturzenbaum, Course director, MSc Biomedical and Molecular Science Research, King’s College London.