Name: characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm
Uploaded: 2017-02-28T14:00:00
Description: Watch this Scientific Journal Video about Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm at JoVE.com

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).

1. Screening for Life Stage and Tissue of Interest with Live Imaging
<ol>
	<li>Culture the marine larvae to competency according to previously reported methods6,7,9. Incubate the tubeworm larvae at 5 larvae per mL density with filtered seawater with 10 &#181;M SNARF-1 AM overnight. Cover the container with aluminum foil to protect the fluorescent probe from photo-bleaching.</li>
	<li>Observe the larvae using a dissection micro...

<ol>
	<li>Aizenberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeleton+of+Euplectella+sp.:+structural+hierarchy+from+the+nanoscale+to+the+macroscale.">Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale.</a> Science. 309 (5732), 275-278 (2005).</li><li>de Nooijer, L. J., Toyofuku, T., Oguri, K., Nomaki, H., Kitazato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+distribution+in+foraminifera+determined+by+the+fluorescent+probe+HPTS.">Intracellular pH distribution in foraminifera determined by the fluorescent probe HPTS.</a> Limnol Oceanogr Methods. 6 (11), 610-618 (2008).</li><li>de Nooijer, L. J., Langer, G., Nehrke, G., Bijma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+controls+on+seawater+uptake+and+calcification+in+the+benthic+foraminifer+Ammonia+tepida.">Physiological controls on seawater uptake and calcification in the benthic foraminifer Ammonia tepida.</a> Biogeosciences. 6 (11), 2669-2675 (2009).</li><li>Smith, A. M., Riedi, M. A., Winter, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperate+reefs+in+a+changing+ocean:+skeletal+carbonate+mineralogy+of+serpulids.">Temperate reefs in a changing ocean: skeletal carbonate mineralogy of serpulids.</a> Mar Biol. 160 (9), 1-14 (2013).</li><li>Carpizo-Ituarte, E., Hadfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+metamorphosis+in+the+polychaete+Hydroides+elegans+Haswell+(Serpulidae).">Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae).</a> Biol. Bull. 194 (1), 14 (1998).</li><li>Bryan, P. J., Kreider, J. L., Qian, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Settlement+of+the+serpulid+polychaete+Hydroides+elegans+(Haswell)+on+the+arborescent+bryozoan+Bugula+neritina+(L.):+evidence+of+a+chemically+mediated+relationship.">Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): evidence of a chemically mediated relationship.</a> J Exp Mar Biol Ecol. 220, 171-190 (1998).</li><li>Chan, V. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+compositional+and+ultrastructural+shifts+during+the+development+of+calcareous+tubes+in+the+biofouling+tubeworm,+Hydroides+elegans.">Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans.</a> J. Struct. Biol. 189 (3), 230-237 (2015).</li><li>Dickson, A. G., Goyet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of methods for the analysis of the various parameters of the carbon dioxide system in sea water. Version 2. , (1994).</li><li>Chan, V. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+deposition+of+crystalline+aragonite+in+the+controlled+biomineralization+of+the+calcareous+tubeworm.">Direct deposition of crystalline aragonite in the controlled biomineralization of the calcareous tubeworm.</a> Front Mar Sci. 2, 97 (2015).</li><li>Bond, J., Varley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+flow+cytometry+and+SNARF+to+calibrate+and+measure+intracellular+pH+in+NS0+cells.">Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.</a> Cytometry A. 64, 43-50 (2005).</li><li>Lloyd, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+number+and+crystallographic+contrast+images+with+the+SEM:+a+review+of+backscattered+electron+techniques.">Atomic number and crystallographic contrast images with the SEM: a review of backscattered electron techniques.</a> Mineral Mag. 51, 3-19 (1987).</li><li>Perez-Huerta, A., Dauphin, Y., Cuif, J. P., Cusack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+electron+backscatter+diffraction+(EBSD)+data+from+calcite+biominerals+in+recent+gastropod+shells.">High resolution electron backscatter diffraction (EBSD) data from calcite biominerals in recent gastropod shells.</a> Micron. 42 (3), 246-251 (2011).</li><li>Bandli, B. R., Gunter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+backscatter+diffraction+from+unpolished+particulate+specimens:+examples+of+particle+identification+and+application+to+inhalable+mineral+particulate+identification.">Electron backscatter diffraction from unpolished particulate specimens: examples of particle identification and application to inhalable mineral particulate identification.</a> Am. Mineral. 97, 1269-1273 (2012).</li><li>Hayat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and techniques of electron microscopy: biological applications. , (2000).</li><li>Wirth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Chem Geo. 261, 217-229 (2009).</li><li>Volkert, C. A., Minor, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+microscopy+and+micromachining.">Focused ion beam microscopy and micromachining.</a> MRS Bull. 32, 389-399 (2007).</li><li>Kudo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtexture+of+larval+shell+of+oyster,+Crassostrea+nippona:+A+FIB-TEM+study.">Microtexture of larval shell of oyster, Crassostrea nippona: A FIB-TEM study.</a> J. Struct. Biol. 169 (1), 1-5 (2009).</li></ol>

Live optical imaging is a useful method for observing cellular events in a multicellular organism. Here, internal pH and calcium ion indicators were used to measure the flux of ions at the mineralization sites. In these regions, active ion pumping is required to elevate pH and Ca2+ concentration to enable calcification2,3. When applying fluorescent molecules to study an organism, it is critical to ensure that the concentration used has negligible toxic...

Biomineralization is a complex series of events, which bridges a suite of cellular activities resulting in the production of exquisitely ordered minerals1. The challenge is to characterize both the dynamic cellular process and the sophisticated mineral structures using a combination of optical and electron microscopy methods. An elevation of intracellular pH favors the formation of CaCO3 crystals, hence, identifying the life stage that has an increased pH reveals the time when calcification is likely to be occurring2,3.
The tubeworms from the family Serpulidae are common calcifiers in the ocean4. It is also a popular invertebrate model for marine research, especially in biofouling5,6. In this study, the process of calcification in mineralizing compartments during biomineralization is observed. The rapid process of metamorphosis includes the emergence of calcium carbonate structures7,8.
We demonstrate how internal pH measurements can be performed on the tubeworm, and how life stages and tissues relevant for calcification can be screened. After the life stage of interest is identified, the tissue responsible for calcification can be characterized at a higher resolution using electron microscopy methods. Using fluorescent microscopy, we determine the time required for calcium carbonate to appear after metamorphic induction. A similar stage of life was subsequently visualized with SEM-EDS for elemental composition distribution, and the deposited mineral was analyzed using two different electron microscopy methods, specifically SEM-EBSD and FIB-TEM.

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characterization of calcification events using live optical and electron microscopy techniques in a marine tubeworm

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.

The following are some observations of the calcification process during the metamorphosis of the tubeworm. Figure 1 shows that the pH values near the collar region is higher than the other tissues after metamorphosis. Figure 2i shows a tubeworm with homogeneous distribution of Ca, suggesting no major calcification events have begun; Figure 2ii shows a tubeworm that has calcified for a longer period, suggesting calcification has gone beyon...

Watch this Scientific Journal Video about Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm at JoVE.com

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

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.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

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