해양 tubeworm 인 라이브 광학 및 전자 현미경 기술을 사용하여 석회화 이벤트의 특성
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
생체 광물 형성 작용 (biomineralization)은 정교하게 정렬 한 무기물의 제조 결과 세포 활성 모음 브릿지 복잡한 일련의 이벤트이다. 문제는 동적 세포 프로세스 및 광학 및 전자 현미경 방법의 조합을 이용하여 복잡한 구조를 모두 무기물을 특성화한다. 세포 내 pH를 상승, 따라서, 증가 된 pH를 갖는 생활 단계를 식별하는 석회화 2, 3이 발생 될 가능성이있는 시간을 계시, CaCO3를 결정의 형성을 선호한다.
가족 Serpulidae에서 tubeworms 바다 4 공통 calcifiers 있습니다. 그것은 특히의 생물 연료 5, 6, 또한 해양 연구를위한 인기있는 무척추 동물 모델이다. 본 연구에서는, 무기질 구획 석회화 과정 두리NG의 생체 광물 형성 작용 (biomineralization)이 관찰된다. 변형의 빠른 처리 탄산 칼슘 구조물 (7, 8)의 출현을 포함한다.
우리는 tubeworm 인에서 수행 할 수있는 방법을 내부 pH를 측정 보여, 삶의 단계 및 석회화에 대한 중요한 조직은 어떻게 선별 할 수있다. 관심의 수명 단계가 식별 된 후에, 석회화에 대한 책임이있는 조직을 전자 현미경 법을 사용하여 더 높은 해상도로 특징 지어 질 수있다. 형광 현미경을 사용하여, 우리는 변성 유도 후 나타나는 탄산 칼슘에 필요한 시간을 결정한다. 삶의 유사한 단계 이후 원소 조성 분포에 대한 SEM-EDS 가시화하고, 증착 된 무기물 개의 상이한 전자 현미경 방법, 구체적으로는 SEM-EBSD 및 FIB-TEM을 사용하여 분석 하였다.
Protocol
Representative Results
Discussion
라이브 광학 영상은 다세포 생물의 세포 이벤트를 관찰하는데 유용한 방법이다. 여기서, 내부 pH 및 칼슘 이온 지표 광물 부위에서의 이온 플럭스를 측정하기 위해 사용되었다. 이 지역에서는 활성 이온 펌프는 석회화 2, 3을 사용하도록 pH와 칼슘 농도를 상승시킬 필요합니다. 유기체 공부 형광 분자를 도포 할 때 사용되는 농도는 무시할만한 독…
Divulgaciones
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 |
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