Caratterizzazione di calcificazione eventi utilizzando dal vivo ottica ed elettronica Microscopia tecniche in 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
Biomineralization è una serie complessa di eventi, quali ponti una serie di attività cellulari conseguente produzione di minerali squisitamente ordinate 1. La sfida è quella di caratterizzare sia il processo cellulare dinamica e le sofisticate strutture minerali utilizzando una combinazione di metodi di microscopia ottica ed elettronica. Un innalzamento del pH intracellulare favorisce la formazione di CaCO 3 cristalli, quindi, identificando la fase vita che ha una maggiore pH rivela momento calcificazioni è probabile che si verificano 2, 3.
I tubicoli della famiglia Serpulidae sono calcifiers comuni nell'oceano 4. È anche un modello invertebrato popolare per la ricerca marina, soprattutto in biofouling 5, 6. In questo studio, il processo di calcificazione nei compartimenti mineralizzanti durisi osserva ng biomineralization. Il rapido processo di metamorfosi prevede l'emergere di strutture di carbonato di calcio 7, 8.
Dimostriamo come interno misurazioni del pH può essere eseguita sul tubeworm, e come le fasi della vita e dei tessuti rilevanti per la calcificazione potrà essere schermato. Dopo la fase di vita di interesse è identificato, il tessuto responsabile calcificazioni può essere caratterizzata con una risoluzione superiore utilizzando metodi di microscopia elettronica. Utilizzando la microscopia a fluorescenza, si determina il tempo necessario per il carbonato di calcio ad apparire dopo l'induzione metamorfica. Una simile fase della vita è stato successivamente visualizzata con SEM-EDS per la distribuzione composizione elementare, e il minerale depositato è stato analizzato utilizzando due diversi metodi di microscopia elettronica, in particolare SEM-EBSD e FIB-TEM.
Protocol
Representative Results
Discussion
imaging ottico diretta è un metodo utile per osservare eventi cellulari in un organismo multicellulare. Qui, sono stati usati indicatori di pH e ioni calcio interni per misurare il flusso di ioni nei siti di mineralizzazione. In queste regioni, pompaggio ione attivo è necessario per elevare il pH e Ca 2+ concentrazione consentire calcificazioni 2, 3. Quando si applica molecole fluorescenti a studiare un organismo, è fondamentale garantire che la co…
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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