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.
Biyomineralizasyon zarif sipariş mineral 1 üretimi ile sonuçlanan hücre faaliyetlerinin bir paketi köprü etkinlikleri kompleks dizisidir. meydan dinamik hücresel süreci ve optik ve elektron mikroskopisi yöntemleri bir arada kullanarak sofistike mineral yapıları hem karakterize etmektir. Hücre içi pH yükselmesi dolayısıyla, artan bir pH değerine sahip döneminde ortaya tanımlayan kalsifikasyon 2, 3 meydana olması muhtemeldir zaman ortaya Caco 3 kristallerin oluşumunu desteklemektedir.
Etti Serpulidae gelen tüp kurtları okyanus 4 ortak calcifiers bulunmaktadır. Özellikle biyolojik kirliliğe 5, 6, aynı zamanda deniz araştırmaları için popüler bir omurgasız modelidir. Bu çalışmada, hidrotermal bölümlerinde kalsifikasyon süreci During Biyomineralizasyon görülmektedir. Metamorfoz hızlı işlem kalsiyum karbonat yapıları 7, 8 çıkmasını içerir.
Biz tubeworm yapılabilir nasıl iç pH ölçümleri göstermek ve yaşam evreleri ve kalsifikasyon hakkında dokular nasıl taranabilir. ilgi hayat aşaması tanımlandıktan sonra, kireçlenme sorumlu doku elektron mikroskopisi yöntemleri kullanılarak daha yüksek bir çözünürlükte karakterize edilebilir. Floresan mikroskobu kullanarak, metamorfik indüksiyondan sonra görünmesi kalsiyum karbonat için gereken zamanı belirler. hayatın benzer bir sahne daha sonra element bileşimi dağıtımı için SEM-EDS ile görüntülendi ve biriken mineral, iki farklı elektron mikroskopi yöntemleri, özellikle SEM-EBSD ve FIB-TEM kullanılarak analiz edildi.
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…
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 |