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 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.
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.
1. Screening for Life Stage and Tissue of Interest with Live Imaging
2. Calibration of Internal pH
3. Analysis of Ratiometric Imaging Data
4. Sample Preservation, Dehydration and Mounting for electron Microscopy
5. Locating a Calcium Rich Region Using SEM-EDS
6. Identifying Crystallographic Information of Calcium Rich Regions Using SEM-EBSD
7. TEM Sample Preparation Using FIB-SEM
8. Obtaining Selected Area Diffraction Pattern on a TEM
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 beyond the time point of interest; Figure 2iii shows a tubeworm with the calcification stage of interest, which was selected for further analysis to understand the tissue responsible for mineralization. Figure 3 shows the higher pH values are correlated with the higher Ca ion signals from both calcein staining and SEM-EDX mapping. From these observations, the tissue from the life stage of interest was examined at a higher resolution using a more localized technique, SEM-EBSD. Upon the discovery of the mineral phase, Figure 4 shows the application of focused ion beam to lift out the material of interest for TEM and selected area diffraction analysis to confirm the crystallinity of the mineral.
Figure 1: Intracellular pH mapping of the tubeworm (from Chan et al. 2015). At 71 h after IBMX treatment, a tubeworm, Hydroides elegans, which represents a slower rate of metamorphosis demonstrates a great heterogeneity in the intracellular pH distribution. DIC image (top left) and pseudocolorized composite images generated from the grey values at 640 nm divided by grey values at 580 nm (top right) are shown. The calculated grey values from the 640/580 nm ratio is proportional to the intracellular pH. The profiles of the grey values from the composite images, were converted into intracellular pH values over the distance along the longitudinal body axis of the tubeworm (bottom panel). The relationship between intracellular pH and 640/580 nm ratio in emission was established by in vitro calibration (y = 0.30x – 1.47; R2 = 0.934; y, grey value; x, intracellular pH). Regions with intracellular pH above pH 8.5 (in red) are corresponding to the regions where calcified structures were found. Scale bars = 100 µm; c, collar; b, branchial lobes; ant, anteior; post, posterior. Please click here to view a larger version of this figure.
Figure 2: Identifying the life stage of interest using SEM-EDS.
Tubeworms at different time point of metamorphosis were screened to capture the newly calcifying stage for further analysis (i) A day 1 post-metamorphic tubeworm shows homogeneous distribution of Ca signal (ii) A faster growing day 2 post-metamorphic tubeworm shows a short ring of Ca signal (iii) A slower growing day 2 post metamorphic tubeworm shows spots of Ca signal. Please click here to view a larger version of this figure.
Figure 3: Characterization of Ca signals using live microscopy (from Chan et al. 2015). (i-iv), SEM-EDS (v-vii) and SEM-EBSD (viii-x). The distribution of calcium ion as correlated to calcein signal at 31 h and 53 h after metamorphosis of the Hydroides elegans. The DIC images (left panel, 3i and iii) and the calcein signal (left panel, 2ii and iv) are shown. Correlative electron microscopy on the day 2 post-metamorphic Hydroides elegans detected spots of calcified structures, as shown in a lower-voltage (5 kV) SEM image (middle panel, 3v). SEM-EDS analysis mapping, performed at 20 kV, shows a heterogeneous distribution of calcium (middle panel, 3vi, Ca; in yellow). The Ca contents are presented as numbers overlaying a greater surface detail of lower-voltage (5 kV) image of the tubeworm, the Ca contents (mol wt%, decimal points are aligned to show the location of spot analyses) obtained from spot quantification (with SEM-EDS at 20 kV) (middle panel; 3vii). Regions with a Ca content higher than 15 mol wt% are likely to be calcified structures. SEM-EBSD analysis of the calcium rich regions is illuastrated in the right panel of 2viii-x. The calcium rich regions (white box) as found in SEM-EDS results were further analyzed with EBSD (right panel; 3viii) (ii) The circle (right panel; 3ix) indicates the EBSD analysis site; a Kikuchi pattern (right panel; 2x) suggests the site is aragonitic from the interface of EDS/EBSD microanalysis software. c, collar; b, branchial lobes; ant, anterior; post, posterior. Please click here to view a larger version of this figure.
Figure 4: Characterization of crystalline structure using FIB-TEM (from Chan et al. 2015). (i) The region exhibiting a Kikuchi pattern from EBSD was excised to prepare a TEM sample, (ii) the removed surrounding material excised by a focused ion beam (FIB) technique (top view), (iii) the material was lifted using a tungsten probe (side view), (iv) the sample with a final thickness of ~200 nm, which was obtained at a lower voltage. Circled area in (v) were analyzed for the presence of selected-area diffraction pattern in a TEM (vi), showing a single-crystal pattern of aragonite. Please click here to view a larger version of this figure.
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 toxicity and enables the organism to perform in a physiologically relevant way. A lower staining concentration would be less toxic and is usually coupled with a longer staining time10. It is important to keep a low density of larvae during the incubation time, minimizing oxygen deprivation and waste accumulation in the staining period.
Prior to pursuing the FIB/TEM methods, which involves a localized region of interest, it is crucial to screen through the life stage and tissue of interest using lower resolution methods like SEM-EDS and SEM-EBSD. Observing the specimen under backscattered mode enables visualization of relative elemental composition where lighter contrast correlates to heavier atomic weight11. Therefore, SEM-BSE images also provide an estimation of where the heavier elements like calcium ions and osmium stained lipid globules are located. SEM-EDS analysis allows more specific mapping of calcium ions on the samples. However, it does not confirm the presence of crystalline CaCO3. SEM-EBSD allows detection of crystalline structures, although conventional EBSD methods require a polished surface12. This study demonstrates how SEM-EBSD on an unpolished sample can provide useful information. A 20 kV beam current enables greater depth of analysis at the sample surface. Therefore, EBSD is a desirable method to detect the presence of biomineral, especially on small, intact biological samples like marine larvae. Although the unpolished samples would give out false negatives when minerals are not facing the detector at about 70°, seeing a Kikuchi pattern is considered to be unambiguous evidence of a mineral phase at the region of interest13.
Transmission electron microscopy (TEM) allows structural characterization of a wider range of materials14. The focused ion beam (FIB) allows for site specific extraction and preparation of a sample with typical dimensions for TEM analysis15. Therefore, the FIB/TEM technique is a suitable method to analyze newly deposited biomaterials at a localized area. Combined FIB-SEM instruments also allow damage-free observation of a specimen through use of an integrated electron beam column. Tungsten sputter coating of the specimen increases conductivity and limits ion implantation and irradiation damage of the sample16. The use of a finely focused ion beam with a low dwell time enables milling of organic materials. Inside the FIB-SEM, a particular feature of interest can be extracted from a bulk specimen and thinned to electron transparency for TEM analysis17. Due to the high energy of the ion beam, crystalline materials may be rendered amorphous. This may occur when preparing a thin lamella for TEM analysis and can be mitigated using a low accelerating voltage ion beam to remove the amorphous layer.
The authors have nothing to disclose.
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).
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 |