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Blackwell Publishing Ltd New tools for labeling silica in living diatoms Julien Desclés1, Mathieu Vartanian1, Abdeslam El Harrak2, Michelle Quinet1, Nicolas Bremond2, Guillaume Sapriel1, Jérome Bibette2 and Pascal J. Lopez11Laboratoire Biologie Moléculaire des Organismes Photosynthétiques CNRS UMR-8186, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France; 2Laboratoire Colloïdes et Matériaux Divisés, ESPCI, 10 rue Vauquelin, 75005 Paris, France Author for correspondence: • Silicon biomineralization is a widespread mechanism found in several kingdoms Pascal J. Lopez that concerns both unicellular and multicellular organisms. As a result of genomic Tel: +33 1 4432 3535 and molecular tools, diatoms have emerged as a good model for biomineralization studies Fax: +33 1 4432 3935 and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited.
Received: 13 July 2007 • Here, new probes were developed specifically to label the pre-existing or the newly Accepted: 2 October 2007 synthesized silica frustule of several diatoms species.
• It is shown that the LysoTracker Yellow HCK-123, which can be used to visualizesilica frustules with common filter sets, presents an enhanced signal-to-noise ratioand allows details of the frustules to be imaged without of the use of ionophores. Itis also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells.
• The coupling of labeling procedures might help to address the challenging questionof the process of frustule exocytosis.
Key words: 3D-imaging, biomineralization, diatoms, exocytosis, nanopattern.
New Phytologist (2008) 177: 822–829
The Authors (2007). Journal compilation New Phytologist (2007)
Diatoms are unicellular eukaryotic algae able to create aesthetic cell walls. They comprise two valves that fit together Silicon biomineralization is a widespread biological process much like a Petri dish and its lid, and which are made of that concerns a large number of organisms, ranging from animals amorphous silica that present species-specific patterns. Owing to higher plants and protists (Epstein, 1999; Ma, 2003; to the extensive variety of these natural structures, understanding Neumann, 2003; Wilt, 2005; Coradin et al., 2006). In the diatom silicification has recently attracted more attention from marine environments, even if this process is found in many chemists, materials scientist and developmental biologists different lineages, some major groups dominate: the siliceous (Coradin & Lopez, 2003; Wilt, 2005). However, studies of sponges, the diatoms, the radiolarians and the silicoflagellates.
diatom cell biology have been hampered in the past by the Interestingly, our current knowledge on the diversity of lack of molecular and cellular tools. This situation is now organisms capable of producing siliceous skeletons is increasing changing following the emergence of new model species for as a result of the discovery of new species (Yoshida et al., 2006) which the number of molecular tools is increasing (Montsant or better phylogeny analyses (Hoppenrath & Leander, 2006).
et al., 2005; Poulsen & Kroger, 2005; Poulsen et al., 2006) However, diatoms occupy a special place among these organisms and for which full-genome sequences are av because they play major ecological roles in carbon and silicon genome.jgi-psf.org/Phatr2/Phatr2.home.html; Armbrust et al., biochemical cycles (Tréguer et al., 1995; Field et al., 1998; 2004). Nevertheless, our understanding of the silica pattern Yool & Tyrrell, 2003).
formation remains limited because the description of biomaterials
usually requires high-resolution microscopy techniques that mixture was stirred at ambient temperature for 2 h. APS was in involve cleaning and selection procedures.
large excess, with an APS/FITC molar ratio of 200 : 1. Prepara- So far, very few fluorescent tracers to study silicon bio- tions were stable and reactive for a few months, so fresh FITC- mineralization have been described. For diatoms, because the APS was made during the different phases of the experiment.
silica polycondensation process (i.e. frustule formation) occurs Furthermore, to ascertain that the solvent does not have any during cell division inside an intracellular acidic compartment effect on the cell viability and/or the membrane permeability, the (a silica deposition vesicle, SDV), acidotropic molecules were synthesis was performed either in dimethyl sulfoxide (DMSO) shown to accumulate inside the SDV, probably because of the or ethanol (EtOH). The results obtained were the same irrespec- protonation of their side chains, and then become trapped in tive of the solvent used (data not shown). Alternatively, at the end the newly synthesized silica structures. The pioneer works of these incubations, an excess (0.6 mm final) of hexamethyl- used rhodamine-123 (Li et al., 1989; Brzezinski & Conley, disilazane (ABCR) was added to inhibit further polycondensa- 1994) as a staining agent, but its low accumulation efficiency tion of the silanes (Haukka & Root, 1994). For overnight or limited its use. More recently, the LysoSensor Yellow/Blue time course experiments, the LysoTrackers (Invitrogen, Cergy DND-160, a useful pH ratiometric indicator (Diwu et al., Pontoise, France) and FITC-APS were used at 1 µm.
1999; Lin et al., 2001), was also used for the imaging ofdiatom frustules (Shimizu et al., 2001; Hazelaar et al., 2005; Image acquisition and processing Vrieling et al., 2005; Frigeri et al., 2006) and to estimate theSi transport in desmosponges (Schroder et al., 2004).
Images were obtained using a Leica DM-IRB microscope coupled Here, we establish a novel fluorescent dye, the HCK-123, to a Z-stage piezo-controller (Sutter Instrument Company, CA, which is observable in the visible light range and can be used USA) with a 100 W mercury lamp. The objectives used are ×63 directly to follow the formation of silica structures. Moreover, we or ×100 (NA 1.4) oil immersion plan APO. The set of filters also show that fluorescein-5-isothiocyanate (FITC)-silane can be used for HCK-123 and FITC-APS were 485/25 nm excitation combined with acidotropic probes to distinguish pre-existing (Ex) and 535/30 nm emission (Em); for DND-160 we used or newly synthesized diatom-shell patterns. This latest develop- 360/40 nm (Ex) and 535/30 nm (Em), and for the chlorophyll ment is also shown to be useful to monitor the exocytosis of the fluorescence signal we used 485/25 nm (Ex) and 675/50 nm silica frustule, a process that has hardly been addressed before.
(Em). For all these sets the same beam splitter, 86 003 bs(Chroma Technology Corp, Rockingham, VT, USA), was used.
The stacks were analyzed using MetaMorph software (Molecular Materials and Methods
Devices Corporation, CA, USA). All the images presented are3D reconstructions. However, in some cases, before the recon- Culture and labeling conditions struction, a deconvolution step using the Meinel algorithm (Meinel, The cells used are the marine diatoms Phaeodactylum tricornutum 1986) and color-specific point spread function was performed.
(clone 1090-1a, Culture Collection of Algae at the Universityof Göttingen), Thalassiosira weissflogii (CCMP 1051, Provasoli- Frustules purification and transmission electron Guillard National Center for Culture of Marine Phytoplankton), microscope (TEM) images Cylindrotheca fusiformis (CCMP 343), Ditylum brightwellii(CCMP 359), the Prasinophyceae Prasinococcus (RCC 520, Exponentially growing cells were fixed with formaldehyde (final Roscoff Culture Collection of Marine Phytoplankton), the concentration 0.8%) for 1 h at room temperature and then Coccolithophoridae Pleurochrysis carterae clone AC1 and the washed several times with distilled water. Organic material was marine red alga Rhodella violacea (strain 115–79, University first oxidized by potassium permanganate (final concentration of Göttingen). The algae were cultured in natural sea water 3%) with an excess of H SO and then eliminated with 16% supplemented with Guillard's (F/2) enrichment solution HNO (v/v) and 48% H SO (2 : 1, v/v) for 1 min. The suspen- (Sigma) and vitamins. In all cases, cells were cultured at 16°C sion was neutralized by adding Tris-HCl buffer (1 m, pH 8), under a light : dark regime (16 h : 8 h). The yeast and the then carefully filtrated and washed with ethanol 95% using a bacteria were cultured at 30°C in YPD (yeast extract/peptone/ Millipore membrane filter (0.45 µm HV). A drop of the cleaned dextrose) and at 37°C in Luria broth, respectively.
material was placed on a 300 mesh carbon-coated copper gridand observed with a Philips Tecnai 12 electron microscope.
Results and Discussion
The FITC-silanes were prepared by mixing 0.265 ml of pure(3-amino-propyl)trimethoxysilane (APS) (ABCR GmbH & Co.
Comparison of the HCK-123 with other lysotrackers KG, Karlsruhe, Germany) and 5 µmol of fluorescein isothiocyanateisomer 1 (Sigma-Aldrich, Paris, France) diluted in 5 ml ethanol To extend the number of useful probes for imaging the as a cosolvent (van Blaaderen & Vrij, 1992) and the reaction dynamics of the frustule formation, we tested several probes The Authors (2007). Journal compilation New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 822–829
Fig. 1 Chemical structures of the principal dyes used in this work.
(a) DND-160 (C H N O ), also called PDMPO (for 2-(4-
pyridyl)-5-((4-(2-dimethylaminoethylamino-carbamoyl)methoxy)phenyl)oxazole); (b) HCK-123 (C H N O ); for acidic compartments that have different biochemical andfluorescence properties. We found that both the LysoTrackerRed DND-99 and the LysoSensor Green DND-153 led to astaining of intracellular membrane components, but extremelyweak or no silica labeling, respectively. The LysoSensor Blue Fig. 2 Comparison of Si-labeling using either HCK-123 or DND-160.
DND-167 was thought to be a good tracer because of its (a) Ditylum brightwellii; (b) Thalassiosira weissflogii. Exponentially high quantum efficiency (Lin et al., 2001), but unfortunately growing cells were cultured in the presence of either HCK-123 or the fluorophore quickly bleaches during light illumination, DND-160 for approx. 20 h before imaging. Note that the more preventing its use for 3D-imaging. We then tested a membrane- important ‘noise' that corresponds to accumulation of the acidotropic probes is far less important for HCK-123 (yellow, right) than for DND- permeable probe, LysoTracker Yellow HCK-123 (Fig. 1a), a 160 (cyan, left). The maximum signal intensity of each picture was weakly basic amine that selectively accumulates in cellular normalized to be about the same value (within 1.5-fold). Bars, 10 µm; compartments with low luminal pH (i.e. lysosomes; Van insets, bright field images.
Hoof et al., 2002; Burgdorf et al., 2007). For comparisonanalyses we also used a pyridyl oxazole dye, the LysoSensorYellow/Blue DND-160 (Fig. 1b). We found that much less nigericin, which are polyether ionophores catalyzing K+ (or background fluorescence was obtained with HCK-123 than Na+) : H+ exchanges) (Shimizu et al., 2001); our experiments with DND-160 for all the diatom species tested. This higher show that this recommendation does not apply for the signal-to-noise (S/N) ratio for HCK-123 compared with Lysoprobe HCK-123. We also found that HCK-123 is more DND-160 can be easily appreciated by analyses of the signal stable, allowing several successive acquisitions, and induces intensities (Fig. 2a,b). With our settings, the autofluorescence less bleaching of the chloroplast, which suggests that it is less signals were negligible and the intracellular signals corresponded detrimental to cell integrity. Moreover, HCK-123 can be used essentially to intravesicular accumulation of the dyes (i.e.
with common filter sets (e.g. GFP or FITC), allowing it to be accumulation in large vacuoles). However, such a signal can used with the vast majority of confocal microscopes that are vary according to the species and the physiological state of the not equipped with UV excitation.
cells. For example, for the large D. brightwellii, the intracellular Altogether, our results prove that different acidotropic pH DND-160 fluorescence can almost completely mask the probes can be used for silica labeling but that their efficiency newly synthesized frustule (Fig. 2a). For T. weissflogii, the dye depends on the ability of the dye to accumulate and to be sta- HCK-123 accumulates almost exclusively in the newly ble inside acidic compartments. For example, the absence of synthesized silica material (Fig. 2b). To increase the S/N ratio labeling of DND-153 could be expected since its apparent it has been recommended to use ionophores (monensin and pKa was measured to be 7.5 (Lin et al., 2001; Molecular New Phytologist (2008) 177: 822–829
www.newphytologist.org The Authors (2007). Journal compilation New Phytologist (2007)
Probes, Invitrogen, Cergy Pontoise, France), therefore limitingits accumulation inside the SDV of diatoms that was estimatedto be an acidic compartment (Vrieling et al., 1999). Anothercriterion for the application of a Lysoprobe for silica labelingmight be the stability or the quenching of both the protonatedand unprotonated forms upon ‘co-precipitation' within thesilica materials (see later discussion). Nevertheless, we foundthat HCK-123 makes a powerful new probe for live cellimaging of the frustule formation.
Sensitivity of the silica labeling To extend our study of HCK-123 further, we chose importantdiatom model species for genomic and/or biomineralizationstudies. To test the sensitivity of the silica labeling, we firstinvestigated two model species that are lightly silicified. Wechose the pennate Cylindrotheca fusiformis, a well-knownspecies in biochemical studies of organic compounds involvedin silica biomineralization (Kroger et al., 2001; Knecht &Wright, 2003; Sumper & Kröger, 2004). For C. fusiformis thesilica wall is limited to a raphe structure and a large numberof girdle bands (Gbs) (Fig. 3a). The raphe is punctuated byrib-like fibulae (their sizes, measured by TEM image analyses,are 102 ± 25 nm; they appear dark in Fig. 3a), which aredense silicified structures separated from one another (by380 ± 134 nm). With our experimental setup, we coulddistinguish the nanometric raphe punctuations as well as themore uniform pattern of the Gbs (Fig. 3a). Another speciesused was P. tricornutum, for which genetic manipulation isroutine (Lopez et al., 2005) and the full-genome sequenceis available. Phaeodactylum tricornutum is polymorphic(cells can be fusiform, oval or triradiate) with only the ovalmorphotype able to synthesize a frustule that is generallyunique to one side of the cell (Lewin et al., 1958; Borowitzka& Volcani, 1978). For this latter species, the silica structurelabeled with HCK-123 resembles a rib with a denser centralnodule and corresponds to the raphe region (Fig. 3b). Finally, Fig. 3 Sensitivity of HCK-123 labeling. (a) Manual drawing of
we investigated the formation of a single Gb in T. weissflogii.
Cylindrotheca fusiformis silica structures. The raphe and the girdle For the clone used, the Gbs are split rings with a width of bands (Gbs) are in gray, and the nonsilicified regions are open. The approx. 600 nm. Approximately 1 h following the addition of 3D-reconstructed image after staining with HCK-123 (yellow) reveals the nano-patterning of the raphe. (b) Manual drawing of HCK-123 to exponentially growing cells, we could visualize Phaeodactylum tricornutum oval cells and transmission electron the formation of a single Gb (Fig. 3c). Altogether our results microscope (TEM) image illustrating the delicate frustule composed demonstrate that HCK-123 can be used to follow the of a central raphe with lateral striae. Fluorescent frustules are found formation of reasonably dense silica structures.
only for the oval morphotype. (c) Manual drawing of a complete frustule of Thalassiosira weissflogii; the fluorescent image reveals the split rings of two newly divided cells. Note the splits that are nearly Coprecipitation of lysotrackers with newly synthesized 180° apart: the tongue-like sections (ligula). The components of the frustule are the epivalve (E) and the hypovalve (H) that are maintained together by the Gbs. Bars, 5 µm (epifluorescence), To ascertain that the novel silica tracer, the Yellow HCK-123, 200 nm (TEM).
was incorporated within the silica matrix per se, preparationsof biomaterials were made. Thalassiosira weissflogii cells were one generation occurred during the labeling period. To purify first labeled overnight in the presence of either DND-160 or organic-free silica frustules, cells were first oxidized by potassium HCK-123. Since the generation time of the cells was c. 12 h, permanganate with an excess of H SO and then treated with and because labeling was performed overnight, usually only a mixture of strong acids for 1 min (see the ‘Materials and The Authors (2007). Journal compilation New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 822–829
Fig. 4 Strategies followed to label silica frustules. To demonstrate that both DND-160 (cyan) and HCK-123 (yellow) are incorporated and
codeposited within newly synthesized silica structures, frustules from overnight cultures were purified. Alternatively, purified frustules could then
be stained by incubation with FITC-APS (green). The arrow shows a partial frustule that is revealed only after fluorescein-5-isothiocyanate (FITC)
labeling. Bars, 5 µm.
Methods' section). After neutralization, fluorescent images of subsequent condensation of silicic acid in alcoholic solutions, the purified materials confirmed that both LysoSensors were as described in Stoëber et al. (1968), were labeled with selectively incorporated and codeposited with Si into newly FITC-APS (Fig. 1c). A much weaker signal was obtained when synthesized frustules (Fig. 4).
the APS coupling moiety was replaced by (3-aminopropyl)dimethylethoxysilane, which possesses only one reactive alkoxygroup coupled to FITC (not shown). These results suggest a In vitro labeling of biogenic silica with FITC-silanes coupling of multiple dyes per surface silanol by polyconden- The need to decipher the silica pattern formation in diatoms sation of FITC-APS. Such a hypothesis is in agreement with has motivated us to explore new kinds of fluorescent dyes that other studies on the use of alkoxysilanes on precipitated silica can label the diatom silica structures ‘independently' of the (de Monredon et al., 2006). Indeed, it is well established that underlying intracellular biological processes. Procedures exist after hydrolysis, alkoxysilanes can condense, either with the to chemically bind a dye molecule to silane coupling agents, surface silanols (here the ones of the frustule) to produce a which can then be used to coat interior or surface silica monolayer coverage via a siloxane anchoring or with itself, particles (van Blaaderen & Vrij, 1992). Such coating involves leading to a polysiloxane network on the surface (Osterholtz the formation of Si-O-Si bonds between the surface silanols & Pohl, 1992).
and the silanes. To the best of our knowledge, the use of silane Finally, we checked that organic-free frustules, either untreated molecules to label biological samples has seldom been reported or prestained after in vivo incorporation of DND-160, (Hodson et al., 1994), but we envisioned that it could be could be labeled with FITC-silane (Fig. 4). Altogether these extended to study the silicate structures of living organisms.
experiments confirmed that FITC-silane can be used specifically The dye FITC was covalently bound to the coupling agent to label either synthetic or biogenic silicates in vitro.
(3-amino-propyl)trimethoxysilane (APS) by an additionreaction of the amine group with the isothiocyanate group.
Use of fluorescent silane to label the silica structure of We then performed kinetics studies, with incubation periods varying between 1, 5 and 30 min and up to 16 h, followed bythree washing steps to increase the S/N ratio. Because incubation We then carried out a series of similar experiments but with time did not significantly influence the signals, we chose the living organisms. We first performed control tests with several shortest incubation of 1 or 5 min. As control, silica beads species that are not silicified: the bacteria Escherichia coli, of 1.2 µm prepared from hydrolysis of alkyl silicates and the baker's yeast Saccharomyces cerevisiae, a Prasinophyceae New Phytologist (2008) 177: 822–829
www.newphytologist.org The Authors (2007). Journal compilation New Phytologist (2007) Prasinococcus, a Coccolithophoridae Pleurochrysis carteraeand the Rhodophyceae Rhodella violacea. Cells cultured inappropriate media were washed before re-suspension in seawater in the presence of FITC-silanes. Except for R. violacea,no staining of intra- or extracellular structures was observed,suggesting that the cells are impermeable to this moleculeand that the silanes did not interact with organic moleculesfrom the outer membrane or the cell wall components (i.e.
glycerophospholipids, lipopolysaccharides (LPS), proteins,glycoproteins and polysacharides). Rhodella violacea showedan important intracellular accumulation of the FITC alone orcoupled to silane, suggesting that these cells are permeable tothe dye molecule (data not shown).
For the diatom T. weissflogii, specific fluorescent signals corresponding to the complete frustules were observed aftertreatment by FITC-silane (Fig. 5a), but no staining wasobtained using FITC or fluorescein alone. Similar results wereobtained for the other diatoms tested: C. fusiformis, S. costatumor D. brightwellii (not shown). It is worth mentioning that thepermeability, and therefore the coating of the biogenic silica,depends on the dye moiety. Indeed, we found for rhodamine-APS a strong intracellular fluorescent signal and a weak stainingof the surface (not shown). This result demonstrates that theefficiency of the silane labeling depends on the dye moiety, andcan be obtained only if the cells are impermeable to the dye.
In addition, careful analyses of the pattern observed for differentcells stained with FITC-silane revealed some heterogeneity.
For example, we often observed a denser structure in the middleof T. weissflogii cells that might correspond to the overlappingregion of the Gbs (Figs 5a, 6a). For T. weissflogii we also foundthat one theca was often more fluorescent than the other (Figs 5a,6a,c). Finally, for C. fusiformis, a preferential labeling of theGbs was reproducibly obtained with the silanes (Fig. 5b). Thevariations of the labeling might reflect some differences in theaccessibility or in the density of the interacting species.
Fig. 5 Labeling of diatom-silica with fluorescein-5-isothiocyanate
Unfortunately, for P. tricornutum, the nonsilicified part of (FITC)-silane. Living cells were treated with FITC-APS for 1 min and then washed before image acquisition. (a) Thalassiosira weissflogii: the extracellular matrix was also stained with FITC-silanes, the complete frustule is stained and the labeling is essentially the even if this signal was approx. two to threefold weaker than same after treatment in SDS/EDTA for 5 min at 95°C. (b) that of the silica frustule (Fig. 5c). However, for the oval cell, Cylindrotheca fusiformis: the labeling corresponds mainly to the Gbs asymmetry in the labeling can exist (Fig. 5a, upper panel left).
region. (c) Phaeodactylum tricornutum: the preferential labeling with Indeed, the two sides of the cell can be more densely labeled FITC-APS that appears as a rib-like structure corresponds to the frustule. However, even if fusiform cells also present a labeling, only than the middle, suggesting that silanes could interact with the silica frustule remains fluorescent after SDS/EDTA treatment. Also some specially localized organic molecules. We also found that note the asymmetric staining of the organic part in some oval cells the FITC-silane signal was reproducibly weaker in fusiform (lower panel). Insets, bright filed images. Bars, 5 µm.
compared with oval cells (Fig. 5c, upper panel). These resultssuggest that FITC-APS can interact with some organic-associated materials, but that this interaction depends on the whereas organic matrix labeling disappeared (Fig. 5c, upper species. To test the specificity of this probe, post-labeled cells and lower panels). The specificity of the frustule-associated were treated with the anionic surfactant sodium dodecyl staining was also confirmed for the other kinds of diatom sulfate (which is commonly used to destabilize electrostatic tested. We interpret that after hydrolysis of the three methanol interactions). After treatment for 5 min in 2% SDS, 100 mm moieties, which will occur in aqueous environments, amino- EDTA at 95°C, the T. weissflogii frustule was still stained, propyl silanes form stable Si-O-Si bridges with accessible surface although with a slightly reduced signal (Fig. 5a); for P. tricor- silanols, leading to stable and resistant staining. Altogether nutum, only the frustule-associated fluorescence remained, our results demonstrate that FITC-APS can be used to visualize The Authors (2007). Journal compilation New Phytologist (2007) www.newphytologist.org New Phytologist (2008) 177: 822–829
Fig. 6 Frustule accessibility can be studied by
dual labeling. Thalassiosira weissflogii cells
were grown in the presence of DND-160
(cyan) and then labeled with fluorescein-
5-isothiocyanate (FITC)-silanes (green).
(a) Before exocytosis, the hypothecae newly
synthesized within the two daughter cells are
stained only by DND-160. (b) After silica
deposition vesicle (SDV) exocytosis, the new
hypothecae present a dual labeling with both
DND-160 and FITC-silanes. Later, the
daughter cells will separate and the newly
formed silica material is clearly visible (c).
Insets correspond to bright fields, and red
images to chlorophyll fluorescence.
the part of the frustules that is accessible from the external The present study provides novel procedures to analyze thesilica structure of living cells with the help of fluorescent Combination of dyes can help to study the release of markers. We have developed the use of a new probe, the LysoTracker Yellow HCK-123, which can be used to follow We believed that the combination of different probes – that the silica formation process in vivo. HCK-123 shows an is, Lysotrackers that stain newly synthesized structures and enhanced S/N ratio and should make a better live probe since FITC-silane that has access only to extracellular materials – it is excitable in the visible range (i.e. less detrimental for the could be used to address the secretion of the valve and Gbs, cell viability). In addition, it opens the possibility of using something that has never before been possible. For this purpose, several fluorescent LysoProbes to study mixed populations or exponentially growing cells were incubated with DND-160 perform combined pulse-chase experiments. In addition, we and then with FITC-APS. For these experiments we found have started the development of another rapid procedure to that DND-160 was a very useful dye since its excitation does analyze the pattern of diatom shells by using FITC-silane not overlap in wavelength with FITC-silane. However, we are coupling agents. In future, since a large number of other currently testing new fluorescent silanes (e.g. Texas red, Alexa amine reactive fluorescent dyes can be coupled to APS, we Fluor) that can be used in combination with the powerful believe that a full panel of fluorescent-silanes could be HCK-123. As illustrated in Fig. 6, we found that, in some T. exploited to address the questions of diatom pattern formation weissflogii cells, the newly synthesized valves are stained only in vivo. Further studies using combinations of different kinds with DND-160 (Fig. 6a); but more frequently in dividing cells of molecular properties and specificities should help to the new hypovalves were labeled with both the DND-160 and address the localization and accessibility of diatom frustules, FITC-silanes (Fig. 6b). After the daughter cells' separation, and hopefully explore the process of frustule exocytosis. Such the dual labeling of the newly synthesized hypothecae is even strategies might also be used to screen for drugs that more visible (Fig. 6c). A likely explanation is that, as we have specifically inhibit the release of the frustule but not its shown that FITC-silanes do not accumulate inside the cells, formation. The aforementioned approaches may also be of the newly synthesized material which is still inside the SDV is interest in the study of silicon biomineralization in other not accessible to FITC-silanes. Upon release, this new silica unicellular or multicellular organisms.
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Women, Mining and An anthology edited by Ingrid Macdonald and Claire Rowland Oxfam Community Aid AbroadNovember 2002 National Office 156 George Street FitzroyVictoria, Australia 3065Telephone: +61 3 9289 9444ABN 18 055 208 636 Website: www.caa.org.auEmail: Oxfam Community Aid Abroad is affiliated with the following organisations:The Australian Council for Overseas Aid (ACFOA), whose code of ethics we are bound by (for a copy of thecode contact the national office); Oxfam International, whose constitution and code of conduct we arebound by; and the Refugee Council of Australia.