Pracownia Mikroskopii Elektronowej Skaningowej

Laboratory of Scanning Electron Microscopy

Uniwersytet Śląski Wydział Biologii i Ochrony Środowiska

University of Silesia Faculty of Biology and Environmental Protection

ul. Jagiellońska 28 40-032 Katowice

tel: (+48 32) 200 93 74 e-mail: [email protected]

www.semlab.us.edu.pl

SCANNING ELECTRON MICROSCOPY in BIOLOGY

Jagna Karcz

Katowice, 2009

I. SEM specimen preparation

FIXATION – GA (glutaraldehyde)

WASHING – phosphate buffer

POST-FIXATION – osmium tetroxide

DEHYDRATION – ethanol or acetone solutions

CRITICAL POINT DRYING (CPD)

COATING

VIEWING SPECIMENS in SEM and IMAGE ANALYSIS

2

Scanning electron mocroscopy (SEM) has many and varied applications in life sciences, earth sciences, and in electrical and mechanical engineering. Scanning electron microscopes produce images of specimen surfaces, so thickness is unimportant, also sample size is only limited by the size of the microscope specimen chamber. However, as with TEM, the vacuum system required by conventional SEMs necessitates removal of any water or volatile component from the specimen.The sample must also be electrically conductive. However, new developments in SEM design are the Low Vacuum and Environmental SEM which maintain the specimen chamber at low vacuum, enabling hydrated, uncoated samples to be imaged.

1. Fixation Biological specimens prepared for SEM are usually chemically fixed. The fixation technique is designed to preserve the cellular structure, so that the scientist can view the specimen in a form as close as possible to that of a living plant or animal structure. Various chemical processes are used to fix the specimen. These chemicals are very dangerous to human health and care must be taken when dealing with them. Primary fixation in a buffered glutaraldehyde solution followed by a post-fixation in osmium tetroxide solution is the most commonly employed means of fixing biological tissue for SEM. Many of researches regularly use the same fixative solutions and schedule for both SEM and TEM. Fixation with glutaraldehyde solutions does not destroy osmotic activity unless the fixation is for a prolonged period or unless tissues are post-fixed in osmium tetroxide.

2. Dehydration Hydrated samples, like most biological and some materials specimens, must first be dehydrated before placing the specimen in the SEM sample chamber. This is typically done by passing the specimens through a graded series of ethanol-water mixtures to 100% ethanol, and then drying the samples by the critical-point method. The most commonly used dehydration fluids are ethanol and acetone. It is reported that ethanol is marginally superior to acetone as a dehydrating medium. It is also mentioned that acetone appears to cause some surface changes to spores, pollen and cells, possibly by dissolving structural lipids.

3. Drying The tissues are introduced into liquid carbon dioxide which is then brought to its critical point, which is the combination of pressure and temperature at which the fluid and gaseous phases exist together without an interface or meniscus. Thus there is no surface tension. It is essential that the specimen is completely dry. The SEM works under a vacuum and for an image to be derived the specimen must be dry, if not the specimen will simply collapse or blow up in the vacuumed chamber. There are several ways to dry the specimens: •

Air-dried: many hard bodies specimens, for example seeds, insects, are dried on capture so once cleaned they can be simply placed into the SEM

•

Critical Point Drying: this complicated process involves simply the replacement of liquid in the cells with gas. This process creates a completely dry specimen with minimal or no cellular distortion.

•

Chemical dehydration: the wet specimen can be put through an alcohol dehydration series which replaces the water with alcohol and then the alcohol is slowly evaporated off leaving a dried specimen. Other dangerous chemicals can be employed to do the same liquid/air replacement and dehydration.

3

4. Mounting Now that the chosen specimen has been fixed, dehydrated and dried the next step is to mount the specimen on an aluminum stub. The stub is often a small, flat, round piece of metal that has a stem – it looks a bit like a flattened mushroom. The basic method of attachment is to glue the specimen or bits of the specimen to the stub which has been covered with double sided sticky tape and a thin layer of foil. The glue is a special silver or carbon conductive glue. We use this to ensure that the specimen (which is not conductive) will be grounded or earthed to the stub, thus ensuring that electron charging of the specimen in the SEM chamber is reduced. The reason for mounting the specimen is to: •

stabilize the specimen in one place for viewing and manoeuvring in the SEM chamber

•

avoid the specimen disappearing when being gold coated

•

reduce the amount of handling of the specimen

5. Gold coating The gold sputter coater is an apparatus that we use to coat the mounted specimens in gold before they go into the SEM. The specimens must be gold coated because most material (but not gold) is transparent to the electron beam used by the SEM. There are two detectors in the SEM chamber which create a signal from electrons bouncing of the gold-coated specimen. These are used to make up an image of the specimen. If the specimen is not finely covered with an electron-opaque substance like gold, the electron beam would travel right through the specimen, creating no image and probably destroying the specimen too. •

Not all the steps mentioned above are necessary to view every specimen. Dry, hard specimens such as plant seeds or minerals may not need fixation or drying. Specimens such as metals may already be electrically conductive and not need coating. Under some conditions, moist biological samples such as leaves and insects can be viewed with no preparation except mounting.

6. Viewing Specimens in the SEM The SEM has a monitor from which we as the operators view the specimen. The image is derived from the detection of excited electrons that are being bounced of the gold specimen at varying speeds and signals. The two detectors pick up the electron signals and via an analog-to-digital conversion process the image is viewed on a computer monitor, captured on black and white negative film or stored as an image on the computer’s harddrive.

4

II. Standard preparation of biological material for SEM analysis 1. Fixation – immerse sample in 3% glutaraldehyde buffered with 0.1 M phosphate buffer at room temperature 0

or 0-4 C (2-4h, max. 24-48h) 2. Washing – rinse tissue with 0.1 M phosphate buffer pH=7.2 - (3 x 10min.) 3. Post-fixation – immerse sample in 1-2% osmium tetroxide in 0.1 M phosphate buffer pH=7.2 (2-4h) at room temperature and in a light tight container. 1-2% osmium tetroxide solution: (1%) - 0.25g OsO4 in 25 ml 0.1 M phosphate buffer (12.5 ml 0.2M phosphate solution + 12.5 ml distilled water) or 2% osmium tetroxide solution: 0.25g OsO4 in 12.5 ml 0.1 M phosphate buffer (6.25 ml 0.2M phosphate solution + 6.25 ml distilled water) 4. Washing in 0.1 M phosphate buffer pH=7.2 (3 x 10 min.) 5. Dehydration in a graded ethanol or acetone solutions in water – 30%, 50%, 70% (can store tissue in 70% ethanol), 80%, 90%, 96%, 100% for 5-15 min each); 2 x 100% ethanol or acetone (15-30 min each) 6. Critical Point Drying CPD 7. Sample mounting 8. Metal coating 9. Viewing specimens in the SEM

I. 0.2 M phosphate solution:

Solution A: Na2HPO4 · 12 H2O - 35.82 g/500ml or Na2HPO4 · 2H2O -17.8 g/500ml Solution B: NaH2PO4 · 2 H2O - 15.6 g/500ml or NaH2PO4 · H2O - 13.6 g/500ml

II. 0.1 M phosphate buffer pH 7.2: add 36 ml solution A to 14 ml solution B (0.2 M phosphate solution) + 50 ml distilled water

100ml 0.1 M phosphate buffer pH 7.2

III. 3% glutaraldehyde: 50 ml 0.2 M phosphate solution + 12 ml 25% glutaraldehyde + 38ml distilled water

5

III. SEM image analysis Based on SEM examination of plant structure, epidermal and seed surface characters may be grouped into four categories: (1) Cellular arrangement or cellular pattern. (2) the primary sculpture of a surface – shape of cells. (3) Secondary sculpture superimposed on the primary sculpture – relief of outer cell walls. (4) Tertiary sculpture superimposed on the secondary sculpture – epicuticular secretions, i.e. mainly waxes and related substances (Fig. 1). Epidermal characters are only slightly influenced by environmental conditions. Their high microstructural diversity of plant surfaces provides most valuable criteria for the classification of Angiosperms and makes them a valuable diagnostic-taxonomic character.

Epidermal surface characters of plants (Figs. 1, 2): 1. Arrangement of epidermal cells – distribution pattern of different cell types with idioblastic elements such as trichomes, glands and stomata

2. The primary sculpture of a surface: shape of cells The most prominent feature of surface sculpturing is usually the cell shape, particularly the curvature of the outer periclinal wall. Under primary sculpture of a plant surface, the following four groups of microcharacters that influence the superficially visible shape of cells are considered: •

Outline of cells. Either isodiametric or elongated in one direction. Usually cells are superficially tetra- to hexagonal and, in the extreme, between 3- and 30-gonal.

•

Curvature of outer periclinal wall. The curvature is responsible for the often macroscopically visible roughness of a plant surface: cells may be flat, concave or convex. There are many descriptive terms for the frequent convex cell forms (e.g. conical, domed, papillate and there exists a fluent transition to unicellular trichomes. The curvature of outer walls can serve as a good diagnostic character for the different taxonomic levels.

•

Anticlinal walls. The superficially visible cell boundaries may be straight, irregularly curved to more or less regularly undulated (sinuated). In descriptive terms, the undulations can be classified into S-, U-, Omega, and V-types. Usually they are of high taxonomic significance.

•

Relief of anticlinal cell boundary. The anticlinal boundary and cell junctions may show particular structures. The boundary can be channelled, raised or depressed.

3. The secondary sculpture: fine relief of the cell wall. The surface of the outer cell wall (surface of cuticle) delikatna kutykularna mikroornamentacja zewnętrznych ścian komórkowych •

Cuticular sculptures. The surface of the outer periclinal and anticlinal cell walls (surface of cuticle) can be smooth or exhibit a micro-ornamentation which, in descriptive terms, could be called striate, reticulate or micro-papillate (verrucose).

•

Secondary wall thickenings can occur in helical to reticulate patterns on the inner side of the outer or inner periclinal walls and on the anticlinal walls of epidermal cells. They are not a surface feature, but they often become superficially visible in connection with shrinkage-deformation of collapsing dead cells; for example frequently in seed coats. The thickenings are usually irregular to helical.

6

4.Tertiary sculpture: epicuticular secretions. Chemically very different epicuticular substances can be secreted by specialized grandular tissue. Epicuticular waxes and related solid lipophilic substances can be secreted by non-specialized epidermal cells. These wax projections may occur in the form of irregular particles such as crystalloid simple rodlets, threads, flakes, massive plates, tubes. Chemically, these secretions consist of alkanes, long-chain alcohols, ketones and esters of long-chain fatty acids. Waxes can be influenced by environmental conditions.

A

B

C

D

E

F

Fig. 1. SEM surface characters of epidermal plant cells (J. Karcz). A) Oenothera parviflora, polygonal cells of the seed coat with straight anticlinal walls and micropapillate secondary sculpture of the outer periclinal walls. B) Oenothera depressa, epidermal testa cells with depressed anticlinal cell boundaries. C) Reynoutria bohemica, abaxial side of leaf surface with stoma and anticlinal undulation of epidermal cell walls. D) Reynoutria japonica, adaxial side of leaf surface. Epidermal cells with irregular cuticular striations on the outer periclinal walls. E) Brassica oleracea, fruit (silique) epidemis with visible epicuticular waxes. F) Brassica campestris, leaf surface with epicuticular wax secretion forming fine platelets and rods. Bars: A-B, 10 µm; C-D, 20 µm; E-F, 5 µm. 7

A

B

C

D

E

F

Fig. 2. SEM micrographs (A, B- Hitachi S3400; C-F – Tesla BS 340). (J. Karcz). A) Mucronothrus nosalis – morphology of mite. B) Chrysolina pardalina – part of the cental intestine. C) Arabidopsis thaliana – trichome stages development. D) Arabidopsis thaliana – mature trichome on the adaxial leaf surface epidermis. E) Chenopodium berlandieri – mature fruit with reticulate surface pattern. F) Zea mays – cross section of the seedling root hair zone. Bars: C- 20 µm; D- 50 µm; E- 500 µm; F100 µm.

8

References:

Barthlott W. 1984. Microstructural features of seed surface. Systematics Association 25: 95-105. Bozolla J.J., Russell L.D. 1999: Electron Microscopy: Principles and Techniques for Biologists. Jones and Bartlett Publishers, Boston. Briarty L.G. 1976: A method for preparing living plant cell walls for scanning electron microscopy. J. Microsc. 94: 191-183. Dykstra M.J., Reuss L.E. 2003: Biological Electron Microscopy: Troubleshooting. Kluwer Academic/Plenum Publishers, New York.

Theory, Techniques, and

Falk RH. 1980. Scanning electron microscopy. Preparation of plant tissues for SEM. SEM Inc., AMF O”Hare (Chicago), IL 60666, USA. Goldstein J., Newbury D.T., Joy D., Lyman C., Echlin P., Lifshin E., Sawyer L., Michael J. 2003: Scanning electron microcsopy and X-ray microanalysis. Kluwer Academic/PlenumPublishers, New York. Hayet MA. 1976. Principles and techniques of scanning electron microscopy. Van Nostrand Reinhold Company Regional Offices, New York. Karcz J. 1996. Scanning electron microscope in carpological studies. Skaningowy mikroskop elektronowy w badaniach karpologicznych. Wiadomości Botaniczne 40(3/4): 55-65. Kittel C. 1992.Wstęp do fizyki ciała stałego, PWN Warszawa. Watkins CD, Sadun A, Marenka S. 1995. Nowoczesne metody przetwarzania obrazu, WNT Warszawa. Internet, www: http://www.emlab.ubc.ca http://www.mos.org/sln/SEM/links.html http://www.itg.uiuc.edu/ms/equipment/microscopes/esem/ www.scimedia.com/chem-ed/imaging/emicrosc.htm http://www.uq.edu.au/nanoworld/links.html http://www.amc.anl.gov/ http://www.mos.org/sln/SEM/links.html http://www.itg.uiuc.edu/ms/equipment/microscopes/esem

9