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

Podsumowanie

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.

Streszczenie

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. 

Wprowadzenie

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. 

Protokół

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

1. 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 H) 6.7 cm x 25.4 cm x 10.2 cm filled with bedding medium prepared with a moist mixture of vermiculite and peat moss (1:1 ratio). Make 4-6 holes of approximately 0.25 cm along the sides of the boxes and on the lid to allow aeration.
2. Gently remove the soil from the nest to uncover the eggs. Wipe the surface of turtle eggs with diluted iodine tincture (1:25,000) to control microbial contamination during incubation. Place the clutches separate from each other, clutch size of painted turtle may vary from 5-9 eggs and place a maximum of 8-9 eggs per box.
   NOTE: Carefully handle the eggs during collection, wiping, labeling and placing inside the boxes. Position and alignment of eggs need to be the same as they were laid, any movement will inhibit embryo development.
3. Manually bury the eggs half in the bedding, cover and place the box inside the incubator set at 30 °C. Incubate the eggs for 10-17 days to obtain the embryonic stages 12, 13, and 18 respectively used in this study. Add distilled water to partially wet the bedding medium every other day to avoid dehydration and to maintain the moisture level for normal development of the embryos.
   NOTE: Incubation and staging embryos are according to a complete developmental table published earlier³⁹.
4. Fix the embryos by making the first cut on one side of the dorsal eggshell and yolk membrane together, vertical to the long axis using pointed scissors. Insert the scissors into the yolk carefully to avoid cutting the embryonic disc. Now cut the lateral side of the egg along the long axis and then cut the other side of the egg along the short axis.
5. Peel open the excised piece with the embryo side up using forceps. Cut the other lateral side of the eggshell and place the excised piece into phosphate buffered saline (PBS at a pH of 7.4).
   NOTE: The turtle egg is filled with highly viscous egg yolk and the dorsal side of the embryo adheres to the eggshell membrane. The eggshell membrane near the embryo changes with the incubation from translucent white to an opaque, chalky white. This allows one to locate the embryo in the center of the long axis and be seen as a dark calcified spot from the exterior^40,41.
6. Use a stereomicroscope to isolate the embryos along with the yolk membrane by peeling them from the eggshell using forceps. Remove extra-embryonic membranes using forceps and micro-scissors. Transfer the embryo using an embryo spoon to fresh PBS in a Petri dish to wash any blood or yolk.
7. Use clear 12-well plates to fix the embryos overnight in 4% paraformaldehyde in PBS at 4 °C or in 2-3% glutaraldehyde in PBS. Place one to three embryos in each well depending on the size of the embryos. Ensure complete infiltration (samples will appear white) for older embryos by extending fixation time for 2-3 days. Rinse embryos 3x with fresh PBS for 5 min each rinse.
   NOTE: Avoid damaging the surface of the embryo by using polystyrene inserts with polyester mesh bottoms for 12-well plates to transfer specimens from one solvent to another.
8. Dehydrate samples using a series of ethanol concentration in distilled water: 30%, 50%, 70%, 80%, 95%, and 100% and treat samples for 1 h in each dehydration solution. Repeat the step with 100% ethanol twice to ensure complete dehydration. If not used immediately, store samples in 70% ethanol at -20 °C for a longer period.
9. Dry embryos using a series of hexamethyldisilazana (HMDS) to 100% ethanol concentration: 1:2, 2:1 and 100%. Leave the samples in each solution for 20 min and keep the Petri dish partially covered during the process.
   NOTE: Carry out all steps involving HMDS in fume hood with necessary personal protection gear as HMDS is highly toxic.
10. Leave the embryos in the final 100% HMDS solution covered completely or partially in a fume hood overnight aiding in evaporation of HMDS, leaving samples ready for mounting and sputter coating. Cover the dish to eliminate dust settling over the samples.
    NOTE: The tissue will appear white after complete drying and partially dried samples will look yellow in color, leave these tissues in the fume hood for a longer time.
11. Choose the size of aluminum stubs and carbon adhesive tape per size of the sample analyzed. Mount the dried samples carefully on a standard aluminum pin stub (12.7 mm x 8 mm) using double stick carbon conductive tape (12 mm).
12. Introduce mounted samples into the chamber of the sputter coater to coat the specimen with a very thin film of gold to eliminate the charge effect. Gold plate the specimens for 60-120 s at a 35 mA sputter.
13. Mount the stubs to corresponding pore-plates by securely fastening the setscrews. Transfer the sample holder into or out of the sample chamber of SEM using the sample exchange tool. Image the samples at high vacuum mode with an accelerating beam voltage of 10 kV and emission current 10 µA.
    NOTE: Always wear gloves while handling samples, sample holders, mounting stubs, and transfer tools to avoid grease contamination from hands to the SEM system.
14. Test alternative techniques listed below to compare the resolution of specimens obtained from above procedure.
    1. Include a post-fixation with 1% osmium tetroxide for 1 h at room temperature after step 1.7.
    2. Partially or completely remove the HMDS at step 1.10 for fast rapid evaporation of HMDS.
    3. Process embryos after step 1.8 using critical point drying (CPD) by following steps 3.3-3.4.

2. Preparing the eggshell for SEM using an air-drying method

1. Collect and incubate the painted turtle (Chrysemys picta) eggs to fix the embryos as specified in steps 1.1-1.7.
2. Save eggshells in distilled water following embryo fixation in step 2.1. Clean the eggshells thoroughly by soaking in distilled water for at least 1 h to eliminate yolk and albumin contamination.
3. Air-dry eggshells after washing on delicate antistatic wipes in the fume hood overnight. Store dried eggshells in clean specimen bottles labeled by number and stage.
4. Mount, sputter coat and image the specimens by following steps specified in steps 1.11-1.13.

3. Critical point drying method for preparing fungal cultures for SEM

1. Establish slide cultures
   1. Prepare potato dextrose agar (PDA) media for fungal cultures: Add 39 g of PDA power in 1 L of distilled water in an Erlenmeyer flask. Mix well by swirling the flask and autoclave the media at 121 °C for 30 min. Allow the media solution to cool and then add the antibiotic chloramphenicol (25 µg/mL) using a sterile micropipette.
      NOTE: After sterilization, the agar solution should be hot and it will not solidify soon. Cool it enough so that it will not inactivate the antibiotics.
   2. Mix it by swirling and pour plates (approximately 10-12 mL of media for each 10 cm Petri dish), carefully stack up and let solidify.
   3. Use a sterile scalpel blade to cut out small blocks of agar about ½ to ¾ of an inch. Remove and place an agar block onto a clean glass microscope slide.
   4. Place the slide in a clean Petri dish to prevent contamination and preserve moisture during incubation.
   5. Raise the slide off the bottom of the Petri dish using a sterile toothpick to create surface tension between the plate and the slide to remove the glass slide without disrupting the delicate growth following incubation.
   6. Use a sterile loop or a needle to transfer some of the fungi from the specimen inoculum to each of the four sides of the agar block on the slide.
   7. Place a clean coverslip on the surface of the agar block following inoculation. Add a few drops of sterile distilled water to the Petri dish around the slide to ensure moisture for the growing fungi.
   8. Seal the plate partially using paraffin film and incubate the plate at 30 ˚C for an appropriate length of time (for Fusarium species incubate for 36 to 48 h).
   9. Remove the slide from the Petri dish and separate the tightly adhered coverslip from the agar block using sterile forceps. Fix the agar blocks in 3% glutaraldehyde in PBS overnight at 4 ˚C.
2. Dehydrate the samples by passing through an ethanol series: 10%, 25%, 50%, 75% and 90% with 15 min per change. Process the samples for final dehydration with two changes in 100% ethanol lasting 30 minutes each to ensure complete saturation.
3. Critical point drying: Place dehydrated samples in the chamber of the CPD apparatus. Seal and cool the chamber by opening the valves to allow liquid CO₂ in and vent ethanol out, until liquid CO₂ completely fills the chamber.
   1. Seal and heat the chamber slowly to achieve a critical point when the chamber pressure exceeds 1000 psi and the temperature exceeds 31 °C, the liquid and gas phase of CO₂ is in equilibrium. Slowly drain the CO₂ from the chamber and the sample as gas to avoid effects of surface tension.
4. Perform mounting, gold plating and imaging the specimens by following steps specified in steps 1.11-1.13.
5. Perform comparative analysis by chemically drying the specimens from step 3.2 using HMDS by following steps 1.9-1.13.

Wyniki

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 ...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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