Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Summary

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Abstract

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial. 

Introduction

Scanning electron microscope (SEM) ultrastructural analysis and intracellular imaging supplement light microscopy for three-dimensional profiling of prokaryotes, plants, and animals. The high spatial resolution of an SEM makes it one of the most versatile and powerful techniques available for the examination of microstructural characteristics of specimens at the nanometer to micrometer scale. Desiccated specimens are resolved to compositional and topographical structures with intense detail, which provides the foundation for developing valid conclusions about functional relationships^{1,2,3,4,5,6,7,8,9}. When interpreting SEM images of biological specimens, it is a great challenge to distinguish between native structures and the artifacts that are created during processing. SEM is generally operated at very high vacuums to avoid any interference from gas molecules affecting the primary, secondary or backscattered electron beams emitted from the sample^10,11. Also, biological materials are susceptible to radiation damage due to their poor or non-conducting properties. It is essential for the specimens loaded into the SEM to be completely dry and free of any organic contaminants to eliminate any possible outgassing in a high vacuum environment^10,11. As biological specimens are mostly composed of water, additional preparative techniques are required to ensure that the native structures are retained.

The resolution obtained is based on optimizing preparation methods specific to specimen types and instrumental parameters utilized. Thus, it is necessary to avoid using generalized processing steps for all tissue types. Some biological specimens will require less stringent processing to preserve their structure while more time and care might be needed for delicate types of samples to avoid the introduction of drying artifacts, such as shrinkage and collapse. Sample preparation is a critical step in SEM imaging; the findings of morphometric studies are remarkably influenced by specimen preparation procedures^12,13. Common preparation steps for many biological samples are fixation, dehydration and coating with a metal such as gold, platinum or palladium to convert their surfaces to be conductive for SEM analysis. The nature and combination of steps used will vary depending on the type of the tissue, and the specific goals of the study. Charge build-up, sensitivity to vacuum and electron beam damage pose problems when processing soft delicate biological samples, necessitating additional processing steps to retain the native structure of the object. Using conventional methods such as osmium tetroxide fixing, and dehydration cause shrinkage and the collapse of delicate tissues^14,15,16,17. The aim of the study is to establish elegant methodologies derived by combining ideas from earlier studies with modifications to prepare and image soft delicate tissues (e.g., reptile embryos, eggshell of painted turtles, and fungal cultures).

Selection of a suitable fixing method is the first most important step for microscopic analysis of biological specimens. Fixing the tissues immediately after isolating from an organism is essential to prevent alteration in their morphology due to decomposition. An effective fixative should terminate cellular processes by permeating the cells quickly and maintaining the effect irreversibly to stabilize the structure of the sample to withstand both subsequent processing steps and examination under the SEM^17,18. Although several chemical and physical fixation methods are known, chemical fixation is more commonly used for biological specimens to avoid any cellular changes due to autolysis, putrefaction, and drying effects. There are numerous fixative chemical formulations discussed in literature^{17,19,20,21,22,23}, fixatives that work by denaturing and coagulating biological macromolecules, and those that fix by covalently cross-linking macromolecules. Alcohols are used as denaturing fixatives that preserve ultrastructure very poorly and are used mostly for light microscopy and not recommended for electron microscopic analysis. Cross-linking fixatives like formaldehyde, glutaraldehyde, and osmium tetroxide create intermolecular and intramolecular crosslinking between macromolecules within the tissues, providing excellent preservation of ultra-structures^11,24,25,26. Biological samples are sensitive to temperature. The temperature at the beginning of fixation is recommended to be 4 °C to reduce the lateral mobility of membrane proteins, to slow the diffusion of intercellular molecules, and to slow the rate of fixation¹¹. The time required for fixing tissues largely depends on the size of the sample and the speed at which the fixative diffuses and reacts with the components of the specimen. An overnight fixation in 4% paraformaldehyde or 3% glutaraldehyde in PBS at 4 °C is the preferred method for SEM analysis of specimens used in this study for their sequential penetrative properties, which allow smaller delicate samples to be processed^{17,18,19,20,27}. A post-fixation step with osmium tetroxide is eliminated not only due to its toxic nature but also found to implement no added advantage to improve image quality for the samples analyzed in this study.

Biological samples contain fluids that interfere with the SEM operation; hence, the samples need to be dried before inserting it in the SEM sample chamber. Once dehydration is ensured, the solvent must be removed from the tissue without creating artifacts into the specimens due to the surface tension/drying. Three different drying methods were commonly used during processing tissues for SEM imaging: air drying, critical point drying, and freeze drying samples^28,29,30,31. Few studies report all three drying methods producing identical results with animal tissue samples^28,29,30,31. A general practice used for smaller specimens are chemical dehydration by ascending concentration series of alcohol and hexamethyldisilazane (HMDS), but larger specimens are dried using a critical point drying (CPD) instrument³². During the drying process, considerable forces formed in small cavities that are passed through the specimen by a liquid/gas interface; this can even lead to a complete collapse of the hollow structures³³. Any deformation occurring due to the treatment could then be mistaken as a native structural feature of the specimen. Thus, the generalized phenomenon for processing should be eliminated and a unique drying process should be standardized for each type of tissue especially when delicate tissue specimens are analyzed.

In several trials conducted using various combination of all the above-mentioned processes, we standardized the methods that can be used for SEM analysis of three delicate tissues: reptile embryos, eggshells of painted turtles, and fungal cultures. Developmental biologists and morphologists describe normal and abnormal morphogenesis during embryo development in representative vertebrate animals. Investigations on gene signaling pathways depend on the morphological description of novel structures. To avoid any abrupt change in the vertebrate embryo structure during SEM analysis, we recommend chemical drying following dehydration. Chemical drying using HMDS is the relatively newest drying method and the advantages include relative quickness, ease of use, lost cost, and the limited expertise and equipment needed⁹. CPD is a commonly used drying technique using passaging CO₂ across the specimens at a specific temperature and pressure. We identified that HMDS is suitable for drying soft delicate tissues and allows larger samples to be processed compared to critical point drying, which caused extensive deformation to embryonic tissues. Several methods have been used to prepare samples for SEM imaging to study the morphological characteristics of fungi³⁴. Fungal specimens are commonly fixed in osmium tetroxide followed by ethanol dehydration and critical point drying, which may provide satisfactory results, although the toxic effects of osmium tetroxide^6,7,35 and losing fungal materials while changing solutions during processing are pronounced disadvantages. The sample preparation technique using air-drying without fixation has also been practiced³⁶ but results in shrunken and collapsed structures, and observation of such specimens can easily be misinterpreted while characterizing the species. Fungal hypha loses its integrity in contact with liquids and an even drying may not be achieved to restore the structure. Due to this effect, freeze-drying is commonly used for drying soft tissues like fungal mycelium. Freeze drying works well for clean materials but the presence of any salts or secretion will obscure surface detail that will be identified only at the SEM viewing stage. We coupled the slide culture method with glutaraldehyde fixing and critical point drying to yield structural details of intact fungal hyphae and spores. Although CPD drying caused shrinkage in embryos, it resulted in well preserved mycelial structures when coupled with glutaraldehyde fixation. The eggshell is of primary importance to the embryo of oviparous animals by not only acting as a protective covering but also providing mechanical stability, permeability to gas and water, and a calcium reserve for the developing embryo. Freshwater turtle eggshells are classified as "rigid" based on their structure, and due to their availability have received significant attention from biologists^{1,2,3,4,5,6,7,37,38}.

We detail simple methods for easy examination of eggshell and shell membranes of painted turtle that can be applied to any rigid eggshell species. Preparation methodologies were evaluated based on resulting image quality and reduced potential artifacts. 

Protocol

NOTE: Painted turtle (Chrysemys picta) eggs used in this study were collected during the nesting season of May through June 2015-16 from Rice Creek Field Station, Oswego New York with permission obtained from the New York State Department of Environmental Conservation (DEC). 1. Chemical drying method to process embryos for SEM Collect turtle eggs from field sites during the nesting season. Prepare the incubation chambers in advance, made of plastic boxes with lids (L x W x…

Representative Results

Figure 1 show scanning electron micrographic analysis of painted turtle (Chrysemys picta) embryos. Painted turtle eggs collected and incubated on a bedding medium, mounted on aluminum stubs following chemical drying were used for SEM imaging (Figure 1A-E). A lateral view of a stage 12 embryo shows the craniofacial structures; maxillary prominence extends beyond the mandibular and limits a well-marked nasal pit medially; five pharyngeal …

Discussion

In our study, different fixation agents, dehydration and drying methods were tested to prepare three different delicate biological samples for SEM imaging: embryos, eggshells, and fungal cultures. SEM is commonly used for surface analysis, so fixative penetration is less concerning, but it must be understood that poorly fixed internal structures will cause inward shrinking or/and collapsed surface structures. Extended fixation time should also be considered for larger tissue samples, replacing the fixative solution a few…

Materials

Agar	Fischer Scientific	S25127A	for slide cultures
Aluminum pin stub	Tedpella	16111	12.7 mm x 8 mm
BD Difco Dehydrated Culture Media: Potato Dextrose Agar	BD 213400	DF0013-17-6	Media for isolation and cultivation of Fungi, yeast and molds
Chloramphenicol	Fischer BioReagents	BP904-100	Antibiotic for media
Coarse Vermiculite	Greenhouse Megastore	SO-VER-12	bedding medium
Clear 12- well plate	Corning	07-201-589	for fixing embryo
Coverslips	Fischer Scientific	S17525B	for slide culture
Critical Point Dryer	Quorum CPD	EMS850	critical point drying
Culture dishes	Fischer Scientific	08 747B	DISH PETRI 100X10MM 12/PK
Ethanol	Fischer Scientific	A406P 4	dehydration agent
Forceps- Aquarius Tweezers	Tedpella	5804	style 4, length 108mm, widh x thickness 0.017 x 0.17 mm
Glutaraldehyde	Fischer Scientific	G151-1	fixative
Gold target for sputter coater	DENTON VACUUM	TAR001-0158	Gold Target, 2.375″ D X .002″
Hexamethyldisilazana	Fischer Scientific	C19479-5000	chemical drying agent
Kim wipes	Kimtech	S-8115	cleaning
Microscope slides	Thermo Scientific	67-762-16	for slide culture
Microscopy Scissors	Tedpella	1327	Double pointed, stainless steel, 100 mm L (3-5/8").
Micro-scissors	Tedpella	1346	Vannas-type, straight, 80mm L
Moria Perforated Embryo Spoon	Fine Science Tools	10370-17	Length 14.5 cm, tip diameter 20 mm, spoon depth 5 mm
Netwell Inserts	Corning	0330B09	15 mm Inserts with 74 µm Mesh Size Polyester Membrane act as handy carriers during specimen processing into different solvents
Paraformaldehyde	Fischer Scientific	T353 500	fixative
Peat moss	Walmart- Miracle Gro	551705263	bedding medium
PELCO tabs double stick carbon conductive tape	Tedpella	5000	12 mm OD
Sputter coater	DENTON VACUUM	DESK V	thin metal coating
SEM	JEOL USA	JEOL JSM 6610LV scanning electron scope	electron microscopy