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Review of Palaeobotany and Palynology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / r ev p a l b o
Morphological, developmental and ultrastructural comparison of Osmunda regalis L. spores with spore mimics Susannah E.M. Moore a,⁎, Nina Gabarayeva b, Alan R. Hemsley a a b
School of Earth and Ocean Sciences, Cardiff University, Cardiff CF10 3YE, Wales, UK Russian Academy of Sciences, Komarov Botanical Institute, St.-Petersburg 197376, Russia
a r t i c l e
i n f o
Article history: Received 25 February 2008 Received in revised form 10 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Osmunda regalis self-assembly ultrastructure morphology development spore mimics
a b s t r a c t A comparison of spores from Osmunda regalis L. and polystyrene-based spore mimics has been undertaken in order to provide insights into the development and formation of relatively primitive fern spores. In recent years, self-assembly experiments have provided different perspectives on the processes involved in pollen and spore wall pattern formation. The spore mimics obtained from the latest experiments closely resemble extant spore types and permit comparison with both immature and mature spores. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The Osmundaceae is composed of three living genera: Osmunda, Todea and Leptopteris, the spores of which have been documented using SEM and TEM (Tryon and Lugardon, 1991). This family, as well as the families Marattiaceae and Ophioglossaceae, is classified as “primitive” by the above authors in regard to the evolutionary level of spore wall structure. This view is shared by others (e.g. van Konijnenburg-van Cittert, 2000). The latter publication also provides a general review of osmundaceous spores through time (from the Triassic to the present), describing the family as homogeneous and stable in respect of its spore characters. A definitive study documenting different developmental stages of extant Osmunda spores was published by Lugardon (1972). This was carried out by the means of TEM, and is supplemented by the SEM study herein. Mature spores of Osmunda regalis range from 50–69 µm in diameter. The sculpture is completely free of a condensed outermost layer which occurs on immature spores. Its tubercles, which are the predominant surface feature, are pronounced as seen both by SEM and LM (Plate I, 1–3). TEM (see, Tryon and Lugardon, 1991) shows a perispore forming a very thin, continuous electron dense layer with the occasional echinate spine occurring where the perispore covers the tubercles. Some of the spines are as long as 1 µm. Exospore wall thickness ranges, in our specimens, from 2 µm over the tubercles to 3.5 µm near the aperture region. These spores are typical of the species (Tryon and Lugardon, 1991). ⁎ Corresponding author. Current address: School of Geography, Earth & Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. E-mail address:
[email protected] (S.E.M. Moore). 0034-6667/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.revpalbo.2008.12.010
In recent years self-assembly experiments and the integration of aspects of colloid chemistry have provided a unique insight into the pattern formation processes involved in pollen and spore wall formation (Gabarayeva and Hemsley, 2006; Hemsley and Gabarayeva, 2006). Spore mimics obtained from recent experiments closely resemble immature spores of Osmunda and by implication may suggest assembly processes applicable to this and other spore types considered primitive. 2. Methods 2.1. Light microscopy Microscopy slides were prepared with fresh spores embedded into Norland Optical Adhesive 61 photopolymer (Techoptics, Tonbridge Kent TN9 1RF England), covered with a cover slip, and cured under UV light (350–380 nm) within 30 min. Norland Optical Adhesive 61 is a clear, colourless, liquid photopolymer with a very good adhesion and solvent resistance, ideal for light microscopy. Samples were documented by digital photography with a Leica DC200 camera and saved as a TIFF file (Tagged Image File Format) for plate preparation. 2.2. Scanning electron microscopy Osmunda regalis spores were scraped from immature and mature sporangia using a razor blade and placed directly onto aluminium stubs. In order to avoid cross contamination, the razor blade was cleaned between each specimen, hands were washed and ready prepared stubs were put into a stub box. Carbon adhesive disks were used (Agar Scientific Ltd.,
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Plate I. Spores of Osmunda regalis L. 1. 2. 3. 4. 5. 6.
SEM micrograph of a mature spore showing its tuberculate sculpture. Scale bar = 15 µm. LM of a mature spore. Scale bar = 10 μm. SEM micrograph showing a detail of the tuberculate sculpture. Note that the perispore is completely formed in mature spores. Scale bar = 4 µm. SEM micrograph of a cluster of immature spores removed from a pale green sporangium. Scale bar = 40 µm. SEM micrograph of a spore which retained a cover of condensed layer on it. Scale bar = 15 µm. Detail of the tuberculate sculpture shown in 5. Note that the cracks are probably artefacts related to the preparation of the spore for SEM observation.
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Stansted CM24 8DA, England) and the specimens coated with gold/ palladium for 1.5 min at a voltage of 15 mA and later viewed using a Phillips XL30 FEG ESEM (Environmental Scanning Electron Microscope) with an average working distance of 10 mm and a voltage of 15 kV–20 kV. The detector used was a secondary electron (SE) detector. Images were obtained digitally with a LEO digital image capture package and saved as TIFF files.
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Sporangia and spores were mounted on a slide and viewed under a light and a fluorescence microscope. 2. Sporangia and spores were fixed using 1:3 glacial acetic acid and ethanol for 24 h and stored in 70% ethanol (Baker et al.1994; Dong et al. 2005). The material was cleared in 5N NaOH for 1.5 h, stained overnight in 0.1% aniline blue in a ready made buffer solution (pH 9) (Micro Essentials, USA) (Martin, 1959). Sporangia and spores were mounted on a slide and viewed under a light and a fluorescence microscope.
2.3. Transmission electron microscopy of spores 3. Results All living material was rinsed with distilled water and directly transferred into 100% acetone and later embedded into Spurr “hard mix” (Glasspool, 2003). The spores were incubated for 24 h in a mixture of acetone/Spurr 50:50, another 24 h in 100% Spurr “hard mix” and finally placed in block moulds to be polymerised at 70 °C for 12 h. The material remained unstained. Sections (between 50 and 100 nm) were taken on a standard ultramicrotome, using a diamond knife, collected on coated copper grids (200 mesh) and viewed using a JEOL 1210 TEM at 80 kV. 2.4. Colloids Polystyrene latex dispersions were used in the production of our mimics since they have some similarities with sporopollenin (e.g. density, refractive index), but more significantly because there is a wide range of literature covering the behaviour of colloidal polystyrene particles, their preparation and behaviour, under differing conditions (e.g. Pieranski, 1983). The mimics utilize a polystyrene latex that is essentially an aqueous suspension of colloidal particles that are typically spherical in shape (polyballs) with a diameter of around 0.2–0.5 µm. Details of the preparation of such a latex are given by Goodwin et al. (1973) and Hemsley et al. (1996, 1998, 2000). The rationale behind the production of mimics has been simple. Some rather basic components (water, styrene, carboxymethylcellulose, NaCl and a variety of hydrophobic hydrocarbons) were selected with the intention, not necessarily of producing structures, but instead, of combining substances similar to those known to be present in the environment of developing sporomorphs (Hemsley et al., 1998). These components map directly, in terms of presumed function, to water (the environment in which sporomorphs develop is essentially an aqueous one) styrene (sporopollenin monomer), carboxymethylcellulose (mucopolysaccharides) and dissolved inorganic solutes and lipids of various forms (salt, cyclohexane and oil). For the mimics used in this study, a proprietary rapeseed oil replaced in part the cyclohexane such that the components were 670 ml distilled water, 50 ml styrene, 10 ml cyclohexane, 30 ml rapeseed oil, 1.25 ml 10% sodium chloride solution and 20 ml 3% ammonium persulphate. Latices were air dried directly upon SEM stubs. Samples were sputter coated with gold/palladium and observed using a Cambridge Instruments Stereoscan 360 SEM (15 kV). 2.5. Transmission electron microscopy of colloids Spore mimics were embedded into small agar blocks (so as not to lose them in the process of preparation). Samples were dehydrated in a series of alcohols and then embedded in a mixture of Epon and Araldite. Ultrathin sections were prepared with a diamond knife and examined in a Hitachi H 600 TEM. 2.6. Staining for callose with aniline blue Fresh Osmunda regalis immature sporangia and spores were stained for callose with aniline blue 1% (aqueous), purchased from Fisher Scientific, England. Two different approaches were pursued: 1. Fresh sporangia and spores were directly incubated in 0.1% aniline blue, and the stain rinsed off with distilled water after 30 min.
3.1. Spore maturation Isolation of individual spores from immature sporangia is difficult, mainly because the spores form large clusters (Plate I, 4). The spores documented below are about 50 µm in diameter and seem to be ‘glued’ together by a condensed layer; their incipient tuberculate sculpture is clearly covered by this layer (Plate I, 5–6). It is unclear as to whether this layer permeates between the spores of the cluster or is present only at the outside of the cluster. Quite a number of spores in these clusters are broken, probably resulting from preparation. Those spores that are broken appear to be thinly walled and hollow (Plate I, 4). TEM documentation of an immature spore, possibly isolated during the embedding process, shows a thick, tuberculate exospore (Plate II, 1). Exospore thickness ranges between 2–3 µm. The latter measurement includes tubercles (Plate II, 1–2). Perispore formation seems to have taken place (Plate II, 2, arrows). The perispore material appears slightly more electron dense than the surrounding resin and lighter than the exospore. It consists of fibrous material and is scattered around the spore. The layer surrounding the spore clusters was not found using TEM (Plate II, 1). It is also noteworthy that none of the spores have a visible trilete mark (Plate I, 4). This is also the case for the individual spores that were sectioned for TEM, where no clear aperture region can be observed (Plate II, 1). The inner wall of the sporangium at this stage of spore development is ribbed and has a smooth surface, on which spherical bodies of about 1 µm in diameter are evenly distributed (Plate II, 3). These particles have previously been interpreted as tapetal residue and both of the terms ‘granules’ and ‘globules’ have been applied (Edwards, 1996: granules; Lugardon, 1981; Tryon and Lugardon, 1991: globules). Here we use the term globules. At a later developmental stage, some spores are isolated but the majority still form large clusters. Average spore diameter is about 50 µm. The layer covering the cluster, as well as the sculpture of individual spores, is still visible (Plate II, 4–5). Trilete marks are now apparent (Plate II, 5). The tuberculate spore sculpture is more pronounced in some of the spores (Plate II, 5), whereas others still seem to be covered by the condensed layer (Plate II, 4). A number of cracks are scattered around the spore sculpture (Plate I, 6). Staining of callose with aniline blue was attempted in order to determine the nature of the condensed layer surrounding the immature spores. Positive staining for callose is auto-fluorescent blue under a fluorescence microscope (Baker et al., 1994; Dong et al., 2005). Before staining, some untreated immature spores were viewed under a fluorescent microscope as a control. They appeared fluorescent blue, without staining. Staining did not seem to affect the spores. No difference was found between stained and unstained spores. Light microscopy did not reveal positive staining in the spores, either. 3.2. Mimics The incorporation of cyclohexane and rapeseed oil into polystyrene latex colloidal systems results in hollow aggregates of polystyrene particles and particle aggregates formed around a central lipid droplet (which we suggest is analogous to the sporocyte).
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Plate II. Spores of Osmunda regalis L. 1. 2. 3. 4. 5. 6.
TEM micrograph showing the wall of an immature spore. Note its thick exospore with tubercles. Scale bar = 4 µm. Detail of the wall shown in 1. Note that the perispore is not completely formed and that finely fibrous material has accumulated towards the exospore (arrows). Scale bar=2 µm. SEM micrograph showing the surface of the inner wall of an immature spore. Note the small spherical bodies on its surface. Scale bar = 10 µm. SEM micrograph showing several maturing spores. Note the slightly protruding trilete mark on two spores (arrow). Scale bar = 20 µm. Sculpture of a maturing spore. Note that the tubercles are free from the condensed layer. Note also the trilete mark (arrow). Scale bar = 10 µm. Spherical mimic constructed of polystyrene (see section ‘Mimics’ in the Results) showing a very similar ‘tuberculate’ sculpture but larger than in the spores of Osmunda regalis. Scale bar = 50 µm.
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Plate II, 6 shows a large polystyrene aggregate (mimic) enclosed by the dried-down mixture of polystyrene, rapeseed oil and smaller aggregates which are the components forming the ‘sculpture’ (Plate III, 1). In some cases the covering of the central lipid droplet is less thick and incomplete which gives rise to porous coatings (Plate III, 2) or holes (ruptures). Some small polystyrene particles and clusters are adherent to the surface of the larger aggregate (Plate III, 1). Internally, as seen using TEM, it is clear that the mimics are hollow and in many cases, where these have been ruptured or are permeable, have been infiltrated with individual latex particles around 0.25 µm in diameter. The mimic walls, which vary considerably in thickness from 0.5 to 5 μm (Plate III, 3–6), consist of a complex mixture of polystyrene particles and particle aggregates. Particle aggregates which protrude from the mimic surface give rise to surface structure (Plate III, 4–6) which ranges from small particle aggregates (which produce tubercles) and chains of individual particles (actually sheets) which produce laminate structures. Depending upon the rapeseed oil concentration at the time of formation of aggregates, they may be hollow or without a central cavity. The majority of smaller aggregates in our Osmunda mimics lack cavities although clearly the larger ones (including those that form the basis of wall accumulation) do have cavities. In either case, aggregates incorporated into the mimic wall combine with particles already present to generate a wall that is largely particulate in construction (see particularly Plate III, 4).
we would expect a chemical reaction between callose and the acid, probably affecting the staining process. The results, however, suggest that this condensed layer does not contain callose. Alternative protocols comprising a pre-fixation in paraformaldehyde, for example, followed by the staining procedure (Jensen, 1962), might lead to a different result. The blue auto-fluorescence of the spores could be explained with the presence of phenolic compounds in the spore walls, which was already demonstrated for Equisetum arvense L. spores (Roshchina et al., 2002). Clustering, both of particles and particle aggregates in the mimic spores is clearly important as it is this mechanism that is largely responsible for their production. In the mimics, the two components most likely to assist in clustering are the carboxymethylcellulose (which will cause flocculations of various kinds) and the cyclohexane/ lipid (droplets) which will cause clustering of lipophillic components at the water/oil interface. The component most like callose in its chemistry is the carboxymethylcellulose although it is generally concluded that its behaviour in mimics is most equivalent to the glycocalyx (Hemsley et al., 2000). The interaction of carboxymethylcellulose in mimic production may be indicative of aspects of the role of callose or a callose-like substance in spore and pollen development, i.e. that its interaction with the sporopollenin precursors may be responsible for the production and form of exospore component aggregates, effectively sculpture but the same may be said for the action of a glycocalyx.
4. Discussion
4.2. Granules/globules
4.1. Clustering
Derived from the tapetum, globules are defined as “small, usually spheroidal, compact bodies of sporopollenin formed within the sporangium at the same time as the exospore either “free” or within the perispore” (Tryon and Lugardon, 1991, p. 633). Sporopolleninous globules have been shown to resist diagenesis and HF preparation and were found in sporangia produced by both plasmodial and secretory types in extant plants (Lugardon, 1981). These globules are believed to be similar to Ubisch bodies (also called orbicules) of spermatophytes (Lugardon, 1981) and in more primitive groups (e.g. Gensel, 1980). We concur with the common view that the tapetum is responsible for the globules such as those seen in Plate II, 3. In Osmunda a plasmodial tapetum exists (summary table in: Parkinson, 1988), probably providing material for perispore formation. During the early stages of development fibrous material is accumulated near the exospore. Tryon and Lugardon (1991) describe, for mature spores, a continuous perispore with echinate elements formed by fascicles of fibrils. The fibrous material seen around the immature spore (Plate II, 2) will probably, at a later stage of development, give rise to the echinate ornamentation. The TEM of the mature spore of Osmunda regalis (see Tryon and Lugardon 1991) does not suggest that globules were incorporated into the outer spore wall towards the end of spore wall formation, as seems to be the case in Drynaria, for example (Tryon and Lugardon, 1991, p. 311). The system used to produce mimics produces a latex which, by its very nature is abundant in particles and particle aggregates, many of which fail to become incorporated into mimic walls and remain free within the reaction vessel (our mimic sporangium). Upon drying of our latex, we find numerous globules and globule-like structures associated with the mimics. Indeed there is a continuous spectrum from small particle aggregates, larger possibly hollow aggregates to the relatively large hollow mimics themselves. Probably what this tells us is that the conditions that give rise to the outer layers of the wall and those that generate globules are the same and what we need to consider in reality is what effect the sporangial wall or the developed spore surface might have in terms of ‘attractiveness’ for free particles and small aggregates. Where these are strongly attracted they will group together as in our mimic walls and those of the developing
Spore clusters of Osmunda regalis appear to be covered by a layer which might consist of condensed locular fluid. The cracks observed on the sculpture of the immature spores (Plate I, 4; Plate II, 4) might reflect the drying locular fluid, suggesting that this is a condensed layer. The layer seems to contain certain components which hold the spores together as a cluster. In plant cells, callose (β-1,3- glucan) is usually utilised as a sealant for plasmodesmata or sieve-plate pores (Strasburger, 1998). Secreted by the tapetum, callose also separates growing pollen (microspores) (Parre and Geitmann, 2005). The latter is known for angiosperm, gnetopsid, conifer and cycad pollen as well as for bryophyte spores (Gabarayeva and Hemsley, 2006). Callose can be broken down enzymatically by callase (β-1,3glucanase), a process which takes place in pollen towards the end of microspore development when they become fully separated from each other (Steiglitz, 1977). The timing of callase secretion is critical for normal pollen development — prematurely induced, it will result in the collapse of the microspores (Bedinger, 1992). One possibility is that the condensed layer around the extant spores illustrated herein is rich in callose, thus forming a cluster. With the secretion of callase, this cluster could break apart nearer to spore maturation resulting in isolated spores that are ready to be dispersed. However, staining for callose with aniline blue did not answer the question as to whether the condensed layer seen around immature spore clusters of Osmunda regalis is made from or contains callose. There might be several explanations for this. Firstly, the discrete layer does not contain callose and therefore no positive staining was achieved. Secondly, the unstained spores are already blue auto-fluorescent, masking any positive staining. We would have expected to observe a positive stain by the means of light microscopy but could not find any difference in the spores before and after aniline blue treatment. Two different staining techniques were attempted. We would have anticipated at least the first protocol, where fresh material was directly dyed with aniline blue, to give a positive result for the presence of callose. The second protocol involved a fixation with ethanol and glacial acid. We are apprehensive about this protocol as
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spores. Where the attraction is less, particles will remain loose within the sporangial fluids (or reaction medium) and could easily end up as debris within the sporangium at maturity. 4.3. Ornament and apertures There are changes in the appearance and pronouncement of the sculpture. Immature Osmunda spores appear clustered (Plate I, 4) and covered by a condensed layer, which is visible in Plate I, 5. Generally, the sculpture is tuberculate and porous, with cracks probably originating from sample preparation for microscopy (Plate I, 6). Later, between immaturity and maturity, the spores start to separate from one another, some becoming isolated (Plate II, 5). The condensed layer is still discernable at this point (Plate II, 4), forming spore clusters. Tubercles and trilete mark appear to be more pronounced (Plate II, 5). At spore maturity the sculpture is free from the condensed layer and the tubercles characteristic of Osmunda regalis spores can be observed (Plate I, 1 and 3). SEM investigation only showed trilete marks in some spores approaching maturity and in mature spores. Although TEM did not reveal an aperture in the immature spore, it is possible that it was sectioned without cutting through the aperture region. In only a very few spores of Osmunda regalis can a trilete mark be observed (Plate II, 4–5). If the trilete marks are not hidden by clustering of the spores, this might imply that in homosporous ferns the tetrad stage in early spore development might not solely be responsible for the formation of the aperture region. For heterosporous lycopsid genera like Selaginella or Isoetes it is suggested that the prominent trilete mark of megaspores is mainly formed from the contact regions resulting from the tetrad formation. It is thought, however, that the trilete mark in Isoetes megaspores, although already visible in immature spores, only becomes fully pronounced towards the end of spore wall formation (pers. comm. Stephen Blackmore, 2005). In Osmunda regalis monolete apertures sometimes occur (van Konijnenburg-van Cittert, 2000), a phenomenon which is not restricted to Osmunda, but has also been observed in “specialised” families (sensu Tryon and Lugardon, 1991) like Pteris (Kremp, 1965; Tryon and Lugardon, 1991). Trilete apertures in ferns are believed to be more primitive than monolete apertures, mainly because they are widely spread among fern families like Osmundaceae, Marattiaceae and Ophioglossaceae (van Konijnenburg-van Cittert, 2000). Trilete marks also occur very early in the fossil record. We suspect that mutations in Pteris or Osmunda could be responsible for occasional aberrant spore development. The exine pattern formation in pollen, for example, is governed by a protein, which, when inactivated, leads to defective pollen wall formation (Paxon-Sowders, et al., 2001). This suggests that aperture formation and expression might also be genetically driven rather than resulting from the contact areas within the tetrad. Mimics develop around cyclohexane/oil droplets and their size is governed by the size of the droplets. If we create conditions in the reaction vessel favouring smaller droplets, we end up with smaller mimics. Mimic size can increase during development by fusion of individual droplets or by accumulation of free lipophillic material from the reaction vessel mixture. The mechanism by which develop-
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ing spores expand is obviously analogous to the second process in which spores must accumulate water and nutritive substances from the surrounding sporangial fluids. The sculptural elements of mimics are, as described above, developed as part of the wall-building process. The sculpture in our mimics is effectively accumulated ‘mini-mimics’; ones that failed to develop on droplets of the cyclohexane/oil emulsion and which, in many cases, have no internal cavities of their own. The implication of this is that the sculpture of real spores could perhaps be developed independently of the basic exospore and incorporated only at later stages of development (rather like the receptor-independent sporopollenin suggested by Rowley and Claugher, 1991). Unfortunately there is evidence to the contrary in that the position of many receptor independent components (in pollen) appears to be tightly governed by the ectexine surface topology (e.g. Rowley et al., 1999) and is supported by our observations of Osmunda herein. The mechanism of sculpture generation in the mimics does not therefore seem to accord with the observation of this process in the living system (Osmunda). Our mimics never produce trilete marks, and that is not really a surprise. They are clearly features of spores which cannot simply be replicated by self-assembly models. Therefore, their formation is probably largely genetically controlled. 5. Conclusions Our observations of the spores of Osmunda regalis have revealed a number of important points as outlined below. • Early stages of spore development seem to lack a trilete mark. • It would appear that our youngest spores are no longer retained in the original tetrads in which they must have been formed. • Spore clusters and individual spores have a condensed coating clearly visible in the early stages of development. It is suggested this is a material that behaves in a similar way to callose. • Osmunda regalis produces free globules within the sporangium during spore development. Comparison with mimics provides a possible explanation for the clustering of spores, a factor that may be linked to the mode of sculpture development. Differential attraction of wall or mimic components results not just in the adoption of sculpture but also the potential adhesion of the spores themselves. The developmental mechanism of mimics also provides a possible explanation for the occurrence of globules as a component of the sporangial content at spore maturity. Nonetheless, although the spores of Osmunda are considered primitive, they have features which are well beyond the predictive capabilities of our relatively simplistic mimics. Acknowledgements Susannah Moore is grateful for having met Bernard Lugardon in Cardiff in November 2006, when he came to examine her PhD thesis “Studies of lower embryophyte spore walls with particular reference to the perispore.” Nina Gabarayeva would like to acknowledge grant RFBR 08-04-00498. The authors thank two anonymous reviewers for their helpful and thought provoking comments and the guest editors for their thorough work.
Plate III. Mimics of the spores of Osmunda regalis L. 1. 2. 3. 4. 5. 6.
Detail of the surface sculpture of a large spherical aggregate as shown in Plate II, 6. Scale bar = 15 µm. SEM image showing a smaller spore wall mimic with tuberculate sculpture and pores. Scale bar = 5 μm. TEM section of a polystyrene mimic. A lipid droplet with a well developed accumulation of particles that show considerable fusion, presumably as a result of their “sticky” condition. Compare with Plate II, 1. This ruptured droplet now lacks content. Scale bar = 1 μm. Detail of the internal structure of some surface accumulations around a cyclohexane/oil droplet. To the top, a more or less rounded accumulation (hemispherical protuberances) shows possible indications of radial chain-like growth and consequent radial porosity. Scale bar = 2 μm. A number of mimics with thin (single particle) walls and tuberculate surface aggregates. Scale bar = 3 μm. Largely monodisperse colloidal particles and particle aggregates accumulated around the surface of a cyclohexane droplet. Note also the surrounding, almost laminar, material where the particles aggregate in a linear radial fashion with respect to the mimic centre. Scale bar = 2 μm.
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