Method Article

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

DOI:

10.3791/55164

⸱

February 28th, 2017

 ,  ,  ,  ,  , 
1Department of Biological Sciences, Clemson University, 2Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 3Advanced Material Research Laboratory (AMRL), Clemson University, 4Swire Institute of Marine Sciences and School of Biological Sciences, The University of Hong Kong

In This Article

Summary

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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

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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

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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 famil....

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Protocol

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1. Screening for Life Stage and Tissue of Interest with Live Imaging

  1. 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 µM SNARF-1 AM overnight. Cover the container with aluminum foil to protect the fluorescent probe from photo-bleaching.
  2. Observe the larvae using a dissection microscope. Competent tubeworm larvae ready for metamorphosis will swim in a forward direction instead of a circular motion.
  3. Transfer the compete....

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Results

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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.......

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Discussion

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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.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Hexamethyldisilazane Electron Microscopy Sciences16700(EM)
Osmium Tetroxide 2% Aqueous SolutionElectron Microscopy Sciences19192
IBMX 3-Isobutyl-1-methylxanthineThermoFisher ScientificPHZ1124
Nigericin, Free AcidThermoFisher ScientificN7143-5MG
35 mm diameter dish, hole size 27 mm, Glass No.0, Non-coatThermoFisher ScientificD110400
5-(and-6)-Carboxy SNARF-1, Acetoxymethyl Ester, AcetateThermoFisher ScientificC-1271
BDH Potassium Chloride, ACS GradeVWRBDH0258-500G
Paraformaldehyde
reagent grade, crystalline		SigmaP6148
1 M Hydrochloric Acid for Volumetric AnalysisWako Pure Chemical Industries, Ltd083-01095
0.05 M Sodium Hydroxide Solution for Volumetric AnalysisWako Pure Chemical Industries, Ltd199-02185
CalceinSigmaC0875
FASWIwaki Co. Ltd.Rei-sea Marine
Mixed Cellulose Ester Membranes; 47 mm dia, 0.45 µmADVANTECA045A047A
ethanolWako Pure Chemical Industries, Ltd051-00476
Artificial seawater for buffersby SOP06 of DOE (1994), cdiac.ornl.gov/ftp/cdiac74/sop06.pdf
Sodium ChlorideWako Pure Chemical Industries, Ltd191-01665
Potassium ChlorideWako Pure Chemical Industries, Ltd163-03545
Magnesium Chloride HexahydrateWako Pure Chemical Industries, Ltd135-00165
Calcium ChlorideWako Pure Chemical Industries, Ltd039-00475
Sodium SulfateWako Pure Chemical Industries, Ltd197-03345
Hydrochloric AcidWako Pure Chemical Industries, Ltd089-08415
2-amino-2-hydroxymethyl-1,3-propanediol (tris)Wako Pure Chemical Industries, Ltd207-06275
2-aminopyridineWako Pure Chemical Industries, Ltd011-02775
Orion 5-star Plus pH meterThermo Scientific
PrpHecT ROSS Micro Combination pH Electrode 8220BNWPThermo Scientific
Axiovision, Version 4.6, Axio Observer Z1Zeiss
ImageJNIH, Bethesda, MD, USA
HRTEM H500Hitachi
SU6600 VPSEMHitachi
NB5000 Focused Ion and Electron Beam (FIB-SEM) systemHitachi 

References

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  1. Aizenberg, J., et al. Skeleton of Euplectella sp.: structural hierarchy from the nanoscale to the macroscale. Science. 309 (5732), 275-278 (2005).
  2. 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. ....

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Tags

Calcium Carbonate CalcificationLive Optical MicroscopyElectron Microscopy TechniquesIntracellular pH ImagingScanning Electron MicroscopyEnergy Dispersive Xray SpectroscopyElectron Backscatter DiffractionTransmission Electron MicroscopyFocused Ion BeamMarine Tubeworm Larvae

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