Caractérisation de calcification Events Utilisation en direct optiques et Electron Microscopy Techniques dans un tubeworm Marine
Summary
We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.
Abstract
Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals.
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.
Introduction
Biominéralisation est une série complexe d'événements, qui relie une série d'activités cellulaires conduisant à la production de minéraux délicieusement commandés 1. Le défi consiste à caractériser à la fois le processus cellulaire dynamique et les structures minérales sophistiquées utilisant une combinaison de méthodes de microscopie optique et électronique. Une élévation du pH intracellulaire favorise la formation de cristaux de CaCO 3, d' où l' identification du stade de développement qui a un pH accru indique le moment où la calcification est susceptible de se produire 2, 3.
Les tubeworms de la famille Serpulidae sont calcifiants communes dans l'océan 4. Il est également un modèle invertébré populaire pour la recherche marine, en particulier dans l' encrassement biologique 5, 6. Dans cette étude, le processus de calcification dans les compartiments de minéralisateurs during biominéralisation est observée. Le processus rapide de la métamorphose comprend l'émergence de structures de carbonate de calcium 7, 8.
Nous montrons comment interne des mesures pH peut être effectuée sur le tubeworm, et comment les étapes de la vie et les tissus concernés pour la calcification peuvent être criblés. Après l'étape de la vie d'intérêt est identifié, le tissu responsable de la calcification peut être caractérisée à une résolution supérieure en utilisant des méthodes de microscopie électronique. En utilisant la microscopie à fluorescence, on détermine le temps nécessaire pour le carbonate de calcium à apparaître après l'induction métamorphique. Une étape de vie similaire a ensuite été visualisée avec SEM-EDS pour la distribution de la composition élémentaire, et le minéral déposé a été analysé à l'aide de deux méthodes de microscopie électronique différents, en particulier SEM-EPCA et FIB-TEM.
Protocol
Representative Results
Discussion
l'imagerie optique en temps réel est un procédé utile pour observer des événements cellulaires dans un organisme multicellulaire. Ici, les indicateurs de pH et des ions calcium internes ont été utilisés pour mesurer le flux d'ions au niveau des sites de minéralisation. Dans ces régions, le pompage de l' ion actif est nécessaire pour élever le pH et la concentration en Ca2 + pour permettre la calcification 2, 3. Lors de l'ap…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to send a big thank you to Clemson Broadcast Productions, audio recording by J. Bright, Narration by A. D. McQuiston, Audio sweetening, K. Murphy, videography by G. Spake, Graphic arts by T. Messervy, Video editing by T. Messervy and E. Rodgers. Technical assistance and scientific advice was inspired by the advice of S. Kawada, S. Kubo, J. Hudson, T. Darroudi, D. Mulwee, H. Qian, Y. W. Lam, M. B. Johnstone, C. Campanati, A. C. Lane, and R. Dineshram. This study was funded by three GRF grants from the HKSAR-RGC (Grant Numbers: 705511P, 705112P, and 17304914).
Materials
| Hexamethyldisilazane | Electron Microscopy Sciences | 16700(EM) | |
| Osmium Tetroxide 2% Aqueous Solution | Electron Microscopy Sciences | 19192 | |
| IBMX 3-Isobutyl-1-methylxanthine | ThermoFisher Scientific | PHZ1124 | |
| Nigericin, Free Acid | ThermoFisher Scientific | N7143-5MG | |
| 35-mm-diam dish, hole size 27 mm, Glass No.0, Non-coat | ThermoFisher Scientific | D110400 | |
| 5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, Acetate | ThermoFisher Scientific | C-1271 | |
| BDH Potassium Chloride, ACS Grade | VWR | BDH0258-500G | |
| Paraformaldehyde reagent grade, crystalline |
Sigma | P6148 | |
| 1 M Hydrochloric Acid for Volumetric Analysis | Wako Pure Chemical Industries, Ltd | 083-01095 | |
| 0.05 M Sodium Hydroxide Solution for Volumetric Analysis | Wako Pure Chemical Industries, Ltd | 199-02185 | |
| Calcein | Sigma | C0875 | |
| FASW | Iwaki Co. Ltd. | Rei-sea Marine | |
| Mixed Cellulose Ester Membranes; 47 mm dia, 0.45 µm | ADVANTEC | A045A047A | |
| ethanol | Wako Pure Chemical Industries, Ltd | 051-00476 | |
| Artificial seawater for buffers | by SOP06 of DOE (1994), cdiac.ornl.gov/ftp/cdiac74/sop06.pdf | ||
| Sodium Chloride | Wako Pure Chemical Industries, Ltd | 191-01665 | |
| Potassium Chloride | Wako Pure Chemical Industries, Ltd | 163-03545 | |
| Magnesium Chloride Hexahydrate | Wako Pure Chemical Industries, Ltd | 135-00165 | |
| Calcium Chloride | Wako Pure Chemical Industries, Ltd | 039-00475 | |
| Sodium Sulfate | Wako Pure Chemical Industries, Ltd | 197-03345 | |
| Hydrochloric Acid | Wako Pure Chemical Industries, Ltd | 089-08415 | |
| 2-amino-2-hydroxymethyl-1,3-propanediol (tris) | Wako Pure Chemical Industries, Ltd | 207-06275 | |
| 2-aminopyridine | Wako Pure Chemical Industries, Ltd | 011-02775 | |
| Orion 5-star Plus pH meter | Thermo Scientific | ||
| PrpHecT ROSS Micro Combination pH Electrode 8220BNWP | Thermo Scientific | ||
| Axiovision, Version 4.6, Axio Observer Z1 | Zeiss | ||
| ImageJ | NIH, Bethesda, MD, USA | ||
| HRTEM H500 | Hitachi | ||
| SU6600 VPSEM | Hitachi | ||
| NB5000 Focused Ion and Electron Beam (FIB-SEM) system | Hitachi |
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