Karakterisering af Forkalkning Events med Live Optiske og elektronmikroskopi teknikker i en Marine Tubeworm
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 er en kompleks serie af begivenheder, der bygger bro en suite af cellulære aktiviteter resulterer i produktion af udsøgt bestilte mineraler 1. Udfordringen er at karakterisere både den dynamiske cellulære processen og de sofistikerede mineralske strukturer ved hjælp af en kombination af optiske og elektronmikroskopi metoder. En højde af intracellulær pH fremmer dannelsen af CaCO 3-krystaller dermed identificerer livstrin der har en forøget pH afslører det tidspunkt, hvor forkalkning kan forventes at blive forekommende 2, 3.
De tubeworms fra familien Serpulidae er almindelige calcifiers i havet 4. Det er også en populær hvirvelløse model for havforskning, især i biologisk forurening 5, 6. I denne undersøgelse processen med forkalkning i mineraliseringsmidler rum during biomineralization overholdes. Den hurtige proces med metamorfose omfatter fremkomsten af calciumcarbonat strukturer 7, 8.
Vi viser, hvordan indre pH-målinger kan udføres på tubeworm, og hvordan livsstadier og væv er relevante for kalcifikation kan screenes. Efter livstrin af interesse er identificeret, kan vævet ansvarlig for forkalkning karakteriseres ved en højere opløsning ved anvendelse af elektronmikroskopi metoder. Ved hjælp af fluorescerende mikroskopi, vi bestemme den nødvendige tid til calciumcarbonat skal vises efter metamorfe induktion. Et tilsvarende tidspunkt i livet blev efterfølgende visualiseret med SEM-EDS for grundstofsammensætning distribution og den deponerede mineral blev analyseret ved hjælp af to forskellige elektronmikroskopi metoder, specielt SEM-EBSD og FIB-TEM.
Protocol
Representative Results
Discussion
Levende optisk afbildning er en nyttig metode til iagttagelse cellulære begivenheder i en multicellulær organisme. Her blev indre pH og calcium ion indikatorer anvendes til at måle strømmen af ioner ved mineraliseringen sites. I disse regioner er aktiv ion pumpning kræves for at ophøje pH og Ca 2+ koncentration for at aktivere forkalkning 2, 3. Ved ansøgning fluorescerende molekyler til at studere en organisme, er det vigtigt at sikre, at…
Divulgations
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 |
References
- Aizenberg, J., et al. Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale. Science. 309 (5732), 275-278 (2005).
- de Nooijer, L. J., Toyofuku, T., Oguri, K., Nomaki, H., Kitazato, H. Intracellular pH distribution in foraminifera determined by the fluorescent probe HPTS. Limnol Oceanogr Methods. 6 (11), 610-618 (2008).
- de Nooijer, L. J., Langer, G., Nehrke, G., Bijma, J. Physiological controls on seawater uptake and calcification in the benthic foraminifer Ammonia tepida. Biogeosciences. 6 (11), 2669-2675 (2009).
- Smith, A. M., Riedi, M. A., Winter, D. J. Temperate reefs in a changing ocean: skeletal carbonate mineralogy of serpulids. Mar Biol. 160 (9), 1-14 (2013).
- Carpizo-Ituarte, E., Hadfield, M. Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae). Biol. Bull. 194 (1), 14 (1998).
- Bryan, P. J., Kreider, J. L., Qian, P. Y. Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): evidence of a chemically mediated relationship. J Exp Mar Biol Ecol. 220, 171-190 (1998).
- Chan, V. B. S., et al. Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans. J. Struct. Biol. 189 (3), 230-237 (2015).
- Dickson, A. G., Goyet, C. . Handbook of methods for the analysis of the various parameters of the carbon dioxide system in sea water. Version 2. , (1994).
- Chan, V. B. S., et al. Direct deposition of crystalline aragonite in the controlled biomineralization of the calcareous tubeworm. Front Mar Sci. 2, 97 (2015).
- Bond, J., Varley, J. Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells. Cytometry A. 64, 43-50 (2005).
- Lloyd, G. E. Atomic number and crystallographic contrast images with the SEM: a review of backscattered electron techniques. Mineral Mag. 51, 3-19 (1987).
- Perez-Huerta, A., Dauphin, Y., Cuif, J. P., Cusack, M. High resolution electron backscatter diffraction (EBSD) data from calcite biominerals in recent gastropod shells. Micron. 42 (3), 246-251 (2011).
- Bandli, B. R., Gunter, M. E. Electron backscatter diffraction from unpolished particulate specimens: examples of particle identification and application to inhalable mineral particulate identification. Am. Mineral. 97, 1269-1273 (2012).
- Hayat, M. A. . Principles and techniques of electron microscopy: biological applications. , (2000).
- Wirth, R. Focused Ion Beam (FIB) combined with SEM and TEM: Advanced analytical tools for studies of chemical composition, microstructure and crystal structure in geomaterials on a nanometre scale. Chem Geo. 261, 217-229 (2009).
- Volkert, C. A., Minor, A. M. Focused ion beam microscopy and micromachining. MRS Bull. 32, 389-399 (2007).
- Kudo, M., et al. Microtexture of larval shell of oyster, Crassostrea nippona: A FIB-TEM study. J. Struct. Biol. 169 (1), 1-5 (2009).
