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In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans

秀丽隐杆线受精期间钙波和其他细胞过程的体内实时成像

Jun Takayama Jun Takayama
MF Masashi Fujita
Shuichi Onami Shuichi Onami

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2205 浏览次数: 10053

评审: Neelanjan BoseManish ChamoliAdler R. Dillman

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Abstract

Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of germline microinjection of a chemical calcium indicator, worm immobilization, live imaging, and image processing that quantifies the calcium fluorescence in the oocyte region moving in the field-of-view during ovulation. This imaging protocol can also be used to image other cellular processes during in vivo fertilization in C. elegans, such as membrane fusion and cytoskeletal dynamics.

Keywords: Fertilization (受精) search Fertilization calcium waves (受精钙波) search Calcium waves (钙波) search Caenorhabditis elegans (秀丽隐杆线虫) search Live imaging (实时成像) search Calcium imaging (钙成像) search Image processing (图像处理) search

Background

Fertilization calcium waves play a pivotal role in egg activation and have been analyzed in various organisms by using in vitro fertilization systems. The nematode Caenorhabditis elegans is amenable to imaging in vivo fertilization because of its translucent body. The fertilization calcium imaging in C. elegans by using a chemical calcium indicator is reported in Samuel et al. (2001). We describe here a protocol modified from the imaging method by applying high-speed spinning disk confocal microscopy and image processing methods that segment the fertilized oocyte region moving in the field-of-view during ovulation. This protocol enables a precise quantitative description of the temporal dynamics of the calcium waves and genetic analysis of the wave pattern.

Materials and Reagents

  1. Glass capillary with filament (NARISHIGE, catalog number: GDC-1 )
  2. 24 x 55 mm cover glass (Matsunami Glass, catalog number: C024551 )
  3. 76 x 26 mm glass slide (Matsunami Glass, catalog number: S011110 )
  4. 18 x 18 mm cover glass (thickness 0.16-0.19 mm; No. 1S/No. 1.5) (Matsunami Glass, special order product)
  5. Micro spatula (e.g., AS ONE, catalog number: 6-524-01 )
  6. 1.5-ml brown tubes (e.g., FUKAEKASEI, WATSON, catalog number: 131-915BR )
  7. Aluminum foil (e.g., UACJ Foil Corporation)
  8. Syringe-driven membrane filter (EMD Millipore, catalog number: SLGS033SS )
  9. 19 (W) x 0.18 (T) mm vinyl tape (3M, catalog number: 35-WHITE-3/4 )
  10. Lint-free wipe (KCWW, Kimwipes, catalog number: S-200 )
  11. Dark chamber (e.g., Ina-optika, model: FB-35BL )
    Note: We make a rectangular hole of approximately 7 x 10.5 cm in the inner lid.
  12. N2 wild-type C. elegans
  13. Agarose gel pad for microinjection
    1. Agarose-LE, Classic Type (Nacalai Tesque, catalog number: 01157-66 )
    2. Or agarose (Thermo Fisher Scientific, Invitrogen, catalog number: 15510-027 )
      Note: This product has been discontinued.
  14. Liquid paraffin (Wako Pure Chemical Industries, catalog number: 164-00476 )
  15. 0.10-µm polystyrene microsphere suspension (Polysciences, catalog number: 00876-15 )
    Notes:
    1. Prepare approximately 500-µl aliquots in 1.5-ml tubes
    2. Store at 4 °C
  16. Serotonin hydrochloride (Sigma-Aldrich, catalog number: H9523 )
  17. Calcium Green-1 dextran, potassium salt, 10,000 MW, anionic (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C3713 )
  18. Nuclease-free water (Promega, catalog number: P1193 )
  19. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  20. Disodium hydrogenphosphate (Na2HPO4) (Wako Pure Chemical Industries, catalog number: 197-02865 )
  21. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  22. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Wako Pure Chemical Industries, catalog number: 131-00405 )
  23. NuSieve GTG agarose (Lonza, catalog number: 50080 )
  24. Vaseline (white petrolatum) (Kozakai Pharmaceutical)
  25. Lanolin (Sigma-Aldrich, catalog number: L7387 )
  26. Paraffin pellets (e.g., Seiwa)
  27. D(+)-glucose (Wako Pure Chemical Industries, catalog number: 041-00595 )
    Note: This product has been discontinued.
  28. 0.2% BSA-fluorescein solution (dissolved in nuclease-free water)
    Note: Prepare immediately before use.
  29. 2.0% gelatin solution
  30. BSA-fluorescein (Thermo Fisher Scientific, Molecular ProbesTM, catalog-number: A23015 )
  31. 100 µM Calcium Green-1 dextran solution (see Recipes)
  32. Sterilized 1 M MgSO4 solution (see Recipes)
  33. M9 buffer (see Recipes)
  34. 10% NuSieve GTG agarose/M9 pad (see Recipes)
  35. VALAP (see Recipes)
  36. Injection recovery solution (see Recipes)
  37. 20 mg/ml serotonin hydrochloride/M9 buffer (see Recipes)
  38. Fluorescence reference slide (see Recipes)

Equipment

  1. Needle puller (NARISHIGE, model: PC-10 )
  2. Microinjector (Eppendorf, model: FemtoJet® Microinjector )
  3. Inverted microscope with manipulator (e.g., Leica Microsystems, model: DMIRB ; NARISHIGE models: MMO-220A , MO-202U , MN-4 )
  4. Hot plate (e.g., IKA, catalog number: C-MAG HP7 )
  5. Microwave oven (e.g., Panasonic, model: NE-EH212 )
    Note: This product has been discontinued.
  6. Microscope (e.g., Leica Microsystems, model: DMRE; Nikon Instruments, model: Ti-E )
  7. 50-ml screw-cap glass bottle (e.g., AS ONE, catalog number: 1-4568-01 )
  8. 100-ml beaker (e.g., ASAHI Glass, AGC, catalog number: 1000BK100 )
  9. 50-ml beaker (e.g., ASAHI Glass, AGC, catalog number: 1000BK50 )
  10. Diamond pen (e.g., Ogura Jewel Industry, model: D-point pen )
  11. High-magnification water-immersion objective lens (e.g., Leica Microsystems, model: HCX PL Apo 63x/1.20 W corr .; Nikon Instruments, model: Plan Apo VC 60XA/1.20 W )
  12. Spinning disk confocal unit (Yokogawa Electric, model: CSU-X1 )
  13. EM-CCD camera (Andor Technology, model: iXon DU-897 )
  14. Ar/Kr laser (IDEX, Melles Griot, model: 643-YB-A01 )
  15. Filter/shutter controller (Ludl Electronic Products, model: Mac 6000 , catalog numbers: 73006081, 73006042, 73006001)
  16. 488-nm laser line filter (IDEX, Semrock, catalog number: LL01-488 )
  17. Dichroic mirror (IDEX, Semrock, catalog number: Di01-T488 )
  18. Long-pass barrier filter (IDEX, Semrock, catalog number: BLP01-488R )
  19. Windows 7 computer for image acquisition
  20. Mac computer for image processing (e.g., ver. 10.6.8 or later)
  21. Interactive pen display (Wacom, model: Cintiq 21UX )

Software

  1. ImageJ (ver. 1.49v or later) (https://imagej.nih.gov/ij/download.html)
  2. Image-acquisition software (e.g., Andor Technology, iQ [ver. 1.10.3 or later] or iQ2 [ver. 2.9.1 or later])

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Takayama, J., Fujita, M. and Onami, S. (2017). In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans. Bio-protocol 7(7): e2205. DOI: 10.21769/BioProtoc.2205.

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分类

细胞生物学 > 细胞成像 > 活细胞成像

发育生物学 > 细胞生长和命运决定 > 卵母细胞

细胞生物学 > 基于细胞的分析方法 > 离子分析

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