Use

Of Scanning Electron Microscope

In Plant Sciences Jai Prakash Keshri CAS in Botany The University of Burdwan email: [email protected]

Introduction Most challenging aspect of botanical research is the understanding of plant structures. Biological importance of these structures lies in the fact that most suitably it explains how an organism in question reacts with its environment. Not only these structures unravel the mystery invisible to naked eye but also it explains the major or minor steps of evolution. Many novel methods & techniques have been developed nowaday to understand cells, tissues, organisms, & whole plants with clarity. Scanning Electron Microscope (SEM), an instrument that has been developed and improved in recent years allowed us to view the surface structures of organisms so clearly never before. It bridges the gap between the whole organisms & the molecules they composed of. Scanning Electron Microscope offers: 1. Remarkable micrographs with three dimensional qualities. This is due to the great depth of field offered which is several hundred times greater than the light microscope. 2. Micrographs with great clarity due to its much higher resolving power, which is about 200-300 nanometers (1 nanometer = 10-9 meters) for a light microscope, while for SEM it is 10-20 nm. 3. A broad magnification range so that large sized specimens (up to 1 cm) could be viewed. 4. Magnification range extending from 15-20 diameters at low end of the range to 50,000 diameters at the high end of range. It is thus possible to produce an electron micrograph of a specimen that is clearly visible to the unaided eye. 5. Sections of biological samples are also clearly viewed through SEM, although its role in viewing natural surfaces is more valued.

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

Methodology Preparation of samples for SEM is most important Detailed techniques are available in standard manuals, but it largely depends upon the natural samples. Workers skills pay much more in this regard. Essentially three steps are prerequisite for final observation. These are: Fixation, Dehydration & Conduction. 1. Fixation: A range of chemical fixatives is being recommended depending on the nature of samples (Robards 1970, Hayat 1970, 1972; Hall1978, Goldstein 1992, Johnson et al.1993) available elsewhere. 2. Dehydration: This is an extremely important step as specimens are exposed to a high vacuum pressure of a range of 10-4to 1O~ torr. A wet specimen with volatile substances may become distorted under such pressure. Chemical drying, airy drying, critical point drying or low temperature freeze-drying is therefore essential for specimens. Dehydration through a graded series of ethanol & acetone is a general practice before the final drying. It is better to place free cells or unicellular plant samples on the poly L lysine coated glass slides or coverslip prior to final dehydration. Critical Point Drying (CPO) is the most important step the specimens be coated with a gold-palladium alloy. It is done by placing the specimens dehydrated in ethanol series or acetone in a drying chamber using liquid carbon dioxide (Anderson 1951). Low temperature freeze-drying using liquid nitrogen at low vacuum has successfully been applied to a variety of plant specimens by Darley & Lott (1973) and Lott (1976). 3. Conduction: Specimens dried by CPO are now mounted on stubs with the help of a double sided sticky tape conducted by silver paint & then finally coated with a gold-palladium alloy. High vacuum is required in order to generate & focus the electron beam in the specimen chambers in this microscope Availability of secondary electrons to the detectors is efficiently permitted in high vacuum for better resolution, depth of field & clarity of images.

Preservation But before proceeding for fixation I suggest the specimens be preserved in 4-5% Formalin solution made from Paraformaldehyde or in Lugol's Iodine (in 100:1 ratio). If the specimens are big enough such as different plant parts, tissue cultures, seeds etc direct fixation may be done but care to be taken for least distortion. If the specimens contain sufficient mucilage use a suitable polysaccharidase that digest the mucilage without causing any harm to the object Specimens from direct cultures yield better results but for specimens that occur scanty in nature or difficult to grow in culture direct collection is the only option. But in these circumstances immediate preservation is necessary. For flagellate members Lugol's Iodine (in 100: 1 ratio) is the better option if not the best

38

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

Prerequisites for a sample to be observed under SEM 1. The sample should be free from any foreign particle, if not treat the sample suitably before fixation. This is very important because it may hinder your observation. 2. The sample should be stable when put under vacuum. 3. The sample should develop minimum surface charges if not nil so that it may remain stable after the exposure of electron beam. 4. After preparation the sample should be able to emit sufficient secondary electrons. Biological samples are commonly soft, poor conductors and have sufficient extra fluids. Therefore they need extra care and need suitable treatments depending on the nature of tissue and the type of study. Various surface compounds such as glycoprotein, cellulose, silica, calcium carbonate, hemicelluloses, cutin, wax, lignin, sporopollenin etc. need suitable treatments for clear understanding of structures under SEM. Paleobotanical plant samples require special treatments depending on the nature and type of fossils (Bajpai 2004). Cleaning by ultrasonic vibrations or chemical treatments is must. On the other hand microfossils or nannofossils in calcareous shales, marl, or chalk could be recovered by treating the samples with a small quantity of hexametaphosphate.

Specimen selection & mounting It depends on: 1. Stage capacity. 2. Type of fixation. 3. Drying procedure use. Keep in mind that it should be smallest enough in order to reduce the artifacts generated in the preparation of the specimen. A suitable base preferably coverslip is needed for suspension of microorganisms, a single cell or a tissue culture sample before going for processing. The specimens are to be mounted on Aluminium or brass stubs with conductive paint or adhesive tape. It should be stable. It should be enough to make good electrical contact with specimen and stub both. If specimens are not large enough avoid direct sticking on the stub. Coverslips or good quality glass slides suitably cut or accommodated on the stub are good options. The simplified schedules of the sample preparations for SEM are given below:

39

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

Living/delicate

tissues

Sample

1 1

Cleaning

Fixation (2-5% Glutaraldehyde depending on the sample in 0.1-0.2 M Cacodylate buffer for four hours at room temperature)

1 1

Wash in Cacodylate buffer

1 % Osmium tetra oxide for four hours at room temperature

l

Proper wash in distilled water

1 DehTa,;o" CPD

1

Sputter coating (Gold or Gold-Palladium alloy)

1

Observation under SEM

40

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

Pollen /Spore Acetolysis of pollen grains (6 - 10 minutes)

1

Rinsing in distilled water (3 minutes for 2 - 3 times)

1 1 1

Dehydration

Centrifuge the sample

Chemical Dehydration (Alcohol series)

1 2, 2 dimethyl propane, in distilled water & acidic medium

1

Acetone: Methanol (1:1) (1 hour)

1 Keep in desiccator for preparation of stub

Mounting and coating

~

Observation under SEM

41

National Workshop on Scanning Electron Microscopy, 26-29 August, 2014

To overcome the difficulties of Conventional Scanning Electron Microscope (CSEM) Environmental Scanning Electron Microscope (ESEM) has came into existence that can image samples in natural state. The superiority of ESEM over CSEM is as follows: 1. Specimens could be viewed without sample preparation & coating. For delicate samples e.g. algae, fungi, bacteria, plant surface structures like trichomes, scales, & glands etc. that have more chances of deformation during dehydration this microscope is more suitable because it need no drying of the specimens. 2. The gas ionization in the sample chamber of this microscope eliminates the charging artifacts of the non conductive samples. 3. Neither the image quality, nor the instrument is degraded due to the outgassing samples or the samples with oil etc as the secondary detectors are almost insensitive to heat.

Implications & Application Plants include an enormous array of organisms starting from minute Cyanobacteria to 400 feet tall Californian Redwoods. Some seaweed called 'kelp' exceeds a height of 60 feet. Plant biologists apply SEM to their respective fields specifically. It is noteworthy that SEM studies sometimes are the exclusive ways of exploration, identification and characterization of plants. It is better to discuss individual plant groups in light of the applications of SEM in different spheres. A. Monerans: Prokaryotic organisms are included in this group. Cyanobacteria, the ancestors of the chloroplast, are the only representatives of this group that link bacteria to the plants. Although TEM proved extremely useful in deciphering the internal structures SEM has made highly significant informations to the surface details. B. Plant Protists: Simple eukaryotes that do not form embryo belong to the kingdom Protista. Most of the algal representatives& their close relatives represent this group. Being a phycologist I find SEM most widely and exclusively applied instrument in many algal groups. To mention a few the following groups have highly appraised applications in this regard: I. Diatoms: These are the unicellular algae made of two parted cell walls called frustules fitting together like face-cream containers or oval and round Tiffin boxes. These frustules are made of silica. The patterns of surface thickenings, ornamentations and other structures are so characteristics that the taxonomic identity of a particular taxon is exclusively dependent on these observations. SEM studies have been found extremely useful in resolving the detailed three dimensional structures with clarity. But before preparing diatoms for SEM it is highly desirable to clean them with specific methods as the organic skin on the frustules may hinder the observations. It is therefore necessary to remove the skin. For detailed methodology readers are advised to consult Sa rode & Kamat (1984) and Round et al. (2007). Both living and fossil forms are studied in this way. It is noteworthy that diatoms play important role in freshwater and marine ecosystem. Moreover fossil diatoms or 42

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

diatomaceous earth are of many use in making dynamite, bricks of blast furnaces and filtration aid in sugar refining processors etc. II. Coccolithophorids: Coccolithophorids are a major group of marine phytoplankton that forms a covering of minute calcite platelets or coccoliths. They were much abundant in late Cretaceous (63-95 million years ago), when very extensive chalk deposits were laid down across much of northern Europe and sites around the world. In fact, the term Cretaceous owes its name to these coccolith chalks (Iatin 'Creta' refers to white earth from the island of Crete). Some black board chalks are derived from such deposits containing coccolith remains. Cretaceous was also marked by a mass extinction of 80% of the coccolithophorid genera as also dinosaurs and ammonites as an impact of massive asteroid or comet off the Yucatan Coast (the famous "KIT" event). Identifications of both extinct and extant coccolithophorids are largely based on coccoliths. Only SEM studies have been found relevant and reliable tool in such identifications. Kleijne (1993) has made significant contributions to the group exclusively on SEM observations. It may be noted that about 1000 species of fossil coccolithophorids are widely used as bioindicators in the oil industries. III. Dinoflagellates, desmids & other unicellular algae: Dinoflagellates (Greek-dinos meaning whirling) constitute an interesting group of marine and freshwater flagellates having a girdle flagellum in the transverse constriction or groove called cingulum and a longitudinal posterior flagellum in the posterior longitudinal groove or sulcus of the body. Although outer most covering of these organisms is the cell membrane, thecal plates of definite number, pattern and orientation occurring below this membrane are highly characteristic and determining feature of individual members. SEM studies have always helped phycologists to make clear three dimensional pictures of these algae. Cleaning the algae with ultrasound, isolation on 8 urn nucleopore filters, & dehydration in a graded acetone series (20-100% in eight steps) give better results. Desmids are also unicellular algae mostly having two semi-cell orientations of vegetative cells and conjugative mode of reproduction. They have characteristic pattern of pores and ornamentations on their body not always discernible through light microscope. It is always required to have the views of individual cells in three dimensions for their identifications. SEM studies have been found useful here also. Some specimens however need treatment with broad range polysaccharidase preparations such as 'glusulase' before fixation. It is better to start study with live specimens but 5% formalin preservation causes no harm for short term delays. Many other unicellular plant protists such as synurophyceans, euglenophytes, scaled chrysophytes and many more little known microscopic forms, have characteristic pattern of surface structures. SEM studies have provided clear light on their identity. These studies have been found extremely useful since these microscopic creatures play significant role in freshwater and marine biomass production, toxicity, climatic changes and global ecology. Hard specimens like oospores, zygospores, cysts, etc need no pretreatment or fixation and specimens may directly be studied on stub with gold plating. But soft parts require specific treatments depending on nature of the parts in question. 43

National

IV.

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

Acritarchs: These are the spherical microfossils of many different origins from various groups of protists. Most paleontologists suspect these structures as sexual or resistant structures. It is almost impossible to investigate these structures without SEM. (B) Bryophytes: Bryophytes are the first land dwellers that adapted land habit as compulsion. They occur in almost all parts of the world but show the greatest diversity in the tropics. Although SEM studies have been done in this group only reluctantly, nature of spores, elaters; peristome may prove useful in understanding the origin and evolution of land plants. These observations certainly will relate evolutionary consequences with the changing environment of the globe through space and time. (e) Vascular plants: Vascular plants constitute land plants that do have true conducting tissues such as xylem & phloem. Psilopsids, Sphenopsids, ferns, gymnosperms& angiosperms are the representative plant groups. Although most studies on SEM have been done on angiosperms, spore features & anatomical details have proved valued applications. I. Sporae dispersae: Spores are uni- or multi-cellular asexual, reproductive or resting bodies, resistant to unfavourable environmental conditions, produced by land plants that can produce new individual when environment is favourable. Spores of all embryo forming plants have a significant amount of sporopollenin (a highly resistant material that resist mechanical breakdown and chemical decomposition). The spores are most common in fossil form and in the air. It is therefore, not surprising that some of the earliest plants fossil records are from spores and many notable features in plant fossil records such as the earliest gymnosperms, angiosperms origins etc. are first detected in pollen record (Willis & McElwain 2002). Besides the three dimensional structures of individual spores the various patterns of ornamentations on different wall layers are unique and easily discernible in SEM. It is better to pretreat fresh pollens & spores with toluene to dissolve fatty acid materials. Freeze microtome is sometimes useful to understand the inner wall layers. Pollen maps are now being prepared for their applications in oil explorations, aeropalynology, iatropalynology (medical aspects such a hay fever, criminology etc.), melittopalynological studies and studies on the pollen allergies. II. Different plant surfaces and structures: Plant surfaces as mentioned earlier are covered by various types of biopolymers such as waxes, cutin etc. Plant waxes have characteristic pattern of deposition. Nature of silicon depositions has been used in distinguishing the genera of angiosperms. Very resistant silica bodies of diverse sizes and shapes allow us to definitely identify cereal grasses and bamboos. Seeds generally have genus specific epidermal cell pattern as well as distinctive sclereid composition (Dickison 2000). SEM observation have significantly contributed and utilized by taxonomists and seed biologists to identify clearly the concerned genera and species to which it belongs. The cuticle is plant equivalent of the fingerprint. Each plant genus has a very distinctive cuticular pattern. It also reflects features such as stomata, papillae and plant glands etc. Epidermal cells have their unique striated or un-striated, pitted or non-pitted, straight or wavy lateral walls. Stomata exhibit diagnostic arrangements. Foliar venation patterns are also distinctive. These surface structures are not always clearly visible in detail even in the best resolutions 44

National

Workshop

on Scanning

Electron

Microscopy,

26-29 August, 2014

of stereo binocular light microscopes. Moreover several type of outgrowths e.g. trichomes; hairs etc. are present on the plant surfaces. Hairs are uniseriate or multiseriate, unicellular or multicellular, branched or unbranched, and glandular or nonglandular. In fern and their allies, these structures are of prime importance since identification of a genus or a group is dependent on those. SEM studies help here also. Three dimensional structures of many internal structures of plant such as sclereids, trichoblasts, trachea, tracheids etc. are being made visible with clarity by SEM. Distinctive type of sclereids, starch grains, crystals of different forms and sizes are the additional features of plants that have much diagnostic value. These studies helped us to explore the origin, interrelationship and diversification of land plants in more reliable way. Distinctive histological features of gymnospermous and angiospermous wood are frequently utilized for their assignment to particular family and genus. Careful examinations of the nature of vessel elements with their perforation plates and thickenings pitting types, cross-field pitting patterns and the occurrence of crystals and/ or other inclusions are necessary in such studies. SEM studies provide additional and concrete features for their identification. Many costly wood samples require such examinations. SEM has been found extremely useful in studying minute fruits because it is difficult to identify them to the plants they belong at the species level. Pericarps of such fruits belonging to certain plants of Apiaceae and Cyperaceae have been studied for useful taxonomic traits (Heywood 1968, Bajpai et a/2002). These informations are extremely useful in pharmacognosy and medical Botany.

Applied Aspects The most interesting aspect of SEM studies is in tracing the adulteration with the help of internal and external plant structures. Many plant products are highly priced. An adulterer may supply a similarly looking substance to saffron that costs around Rs-1 ,00,000/- per K.G. or low quality tea leaves in place of high priced tea samples. Similarly less valuable wood & fibers are substituted for those of higher quality. In forensic science anatomical evidences have always added additional clues in tracing the crime. Many legal questions have been resolved & several criminals have been caught by anatomical & structural evidence from plants. In this respect SEM details have provided more better and reliable support. Forensic Botany in fact developed at its high esteem with the help of this instrument. Identification is extremely important in pharmacognosy, medicine and animal nutrition. SEM has provided extreme resolution for such identifications, archeologists, palaeoethnobotanists, & anthropologists find SEM extremely important in their investigations. Charcoal samples recovered from archeological sites could be identified up to the genus or family level with SEM. It provides evidence of the type of woods selected by ancient people as fuelwood and for construction of their artifacts & other structures. Palaeoethnobotanists imply charcoal assemblages by quantifying the amount of each taxon occurring at a site. In this way the identification of plant taxa as indicator of the erstwhile plant communities might indicate the past environment. It is therefore very easy to develop a chronology of vegetation change of a 45

National Workshop on Scanning Electron Microscopy, 26·29 August, 2014

given region or to adopt a project to revegetate now barren areas or areas where native plants have been dominated or replaced by non-native plants. Anthropologists use this instrument in their own way. Human coprolites give important clues of the food habits of prehistoric man. For example SEM observations of the transverse sections of the plant fragments in the coprolites of the cave sites of Nevada identified the specimen as root fragments of an aquatic angiosperm Sagitta ria (Alismtaceae).

Conclusive Remarks To conclude its justified to say that and understanding plant relations applied fields of the Botanical iatropalynology, melittopalynology, adulteration to food products and in

SEM studies not only have added high values in tracing in taxonomic and phylogenetic perspectives but in the sciences such as oil exploration, aeropalynology, anthropology, pharmacognosy, and also in tracing the forensic sciences.

Further Readings [1] Anderson, TF 1951. Techniques for the preservation of three dimensional structures in preparing specimens for electron microscope. Trans. N. Y. Acad. Sci. ser. II 13: 130-134. [2] Bajpai, U. 2004. Scanning Electron Microscopy and its application in study of plant tissues. !n Vistas in Palaeobotany and Plant Morphology: Evolutionary and Environmental Perspective. Prof. D.O. Pant Memorial Volume (ed. Srivastava, P.C.), U.P. Offest, Lucknow, India. Pp.295-306. [3] Bajpai, U., Ambwani, K. and Saini, D. C. 2002. SEM studies in the fruit morphology of the genus Cyperus (Cyperaceae). Phytomorphology 53: [4] Darly, J. J. and Lott, J. N. A. 1973. Low temperature freeze-drying for the scanning electron microscope using liquid nitrogen at low vaccum. Micron 4: 178-182. [5] Dickison, W.C. 2000. Integrative Plant Anatomy. Academic press, U.S.A. [6] Goldstein, DE 1992. Scanning electron microscopy and X- ray Microanalysis. 2nd ed. Planum press. [7] Hall, J.L. 1978. Electron Microscopy and Cytochemistry of Plant cells. Elsevier/NorthHolland Biomedical Press, Amsterdam-Oxford-NY. [8] Hayat, MA 1970. Principles and techniques of electron microscopy: biological applications. Reinhold Co. NY & London. [9] Hayat, MA 1972. Basic Electron Microscopy Techniques. Reinhold Co. NY & London. [10] Heywood, V. H. 1968. Scanning Electron Microscopy and microcharacters in the fruits of the Umbelliferae-Caucalideae. Proc. Linn. Soc. London 179: 287-289. [11] Jonson, J.E., Griffith, E.M. & Danilatos, G.D. 1993. Microscopy Research an Techniques. 25: 5-6. {12]Kleijne, A. 1993. Morphology, Taxonomy and Distribution of Extant Coccolithophorides (Calcareous Nannoplanctons). Drukkerij FEBO B. V., Javastraat. [13] Robards, A. W. 1970. Electron Microscopy and plant ultrastructure. McGraw Hill, England. 46

National Workshop on Scanning Electron Microscopy, 26-29 August, 2014

[14] Round, F. E., Crawford, R. M. & Mann, D. G. 2007. The diatoms: Biology and morphology of the genera. Cambridge University Press.pp.747. [15]Sarode, P. T. & Kamat, N. D. 1984. Freshwater Diatoms of Maharashtra. Saikripa Prakashan, Aurangabad. [16IWillis, K. J. & McElwain, J. C. 2002. The Evolution of Plants.Oxford, N.Y.

47