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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   

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

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

Note: This protocol is modified from the original method described in Samuel et al. (2001).

  1. Microinjection of chemical calcium indicator dye
    1. Inject 100 µM Calcium Green-1 dextran solution into the distal arm of the anterior gonad of a gravid adult hermaphrodite by using a standard method for C. elegans DNA microinjection (see Evans, 2006). We use a GDC-1 glass capillary with filament pulled by the PC-10 puller in the double-pulling mode. In addition, we use a 2% or 3.5% agarose gel pad on a 24 x 55 mm cover glass for worm immobilization and liquid paraffin to protect the specimen from drying.
      Notes:
      1. Fill about half the gonad as described in Samuel et al. (2001). Injecting too much dye often results in an abnormal gonad shape after recovery.
      2. We always place only one worm per agarose pad for injection to minimize the time that the worm is immobilized and hence minimize the damage.
      3. We typically inject four to six worms in a single set of experiments (e.g., one genotype) and perform two sets of experiments in a day with a 2-h interval. Approximately half of the injected worms can be imaged for each set of experiments.
    2. To recover the injected worm, apply approximately 1 µl of injection recovery solution onto the worm by using a P20 micropipette.
    3. By using a P20 micropipette, suck up the injected worm in the drop of injection recovery solution and transfer it from the agarose pad to a 3.5-cm OP50/NGM plate.
      Note: A single injection procedure from picking to transfer (steps A1-A3) should be completed within approximately 5 min.
    4. Allow the injected worm to recover on the plate in a dark chamber for 4-6 h at room temperature.
    5. Check whether the injected dye is incorporated into the oocytes of the worm by using a stereo fluorescence microscope.
      Note: Sloppy injection can result in leakage and incorporation of the dye into the coelomocytes (Samuel et al., 2001).

  2. Worm preparation and imaging
    Note: The worm-immobilization method is modified from the original method described in Kim et al. (2013).
    1. Melt VALAP at 130 °C on a hot plate.
    2. Prepare a 10% agarose gel pad.
      1. Prepare two double-taped glass slides (Figure 1A). The thickness of the tape stack in this image was 0.36 mm.
      2. Melt the 10% NuSieve GTG agarose/M9 buffer in a microwave oven.
      3. Apply a drop of the melted 10% NuSieve GTG agarose/M9 buffer to a glass slide between the two taped glass slides by using a micro spatula (Figure 1A; Video 1).
      4. Cover the melted agarose drop with another glass slide (Figure 1B; Video 1).
      5. Incubate for at least 30 min at room temperature to solidify the agarose pad. Keep the agarose pad covered by the glass slide until use.


        Figure 1. Sample preparation. A. A drop of 10% agarose is applied to a glass slide between the two double-taped spacers by using a micro spatula. B. A slide glass is placed on the 10% agarose drop. C. A drop of microsphere suspension and a drop of serotonin/M9 solution are applied to the agarose pad on the stage of a stereo microscope. D. A sample ready for imaging, in which the cover glass is stabilized by VALAP on the agarose pad (a non-injected worm is immobilized in this image).

        Video 1. Preparation of a 10% agarose gel pad


    3. Allow the polystyrene microsphere suspension and the serotonin hydrochloride/M9 buffer to warm at room temperature for at least 30 min before use.
    4. Check the shape of the leading oocyte of the injected worm to determine the timing of ovulation by using the stereo fluorescence microscope. The shape of the leading oocyte should appear rectangular, and its nucleus should be in the cortical region distal to the spermatheca (Figure 2).


      Figure 2. Inference of the timing of ovulation on the basis of the oocyte shape. A. An oocyte that appears ready to be ovulated within a few minutes. The oocyte shape is rectangular, the boundary facing the next oocyte looks like a straight line (indicated by the black line), and the nucleus is close to the cortical region distal to the spermatheca. B. An oocyte not ready for imaging. The boundary has an arc-like appearance (indicated by the black line), and the nucleus is not close to the distal cortex. Differential interference contrast images of non-injected wild-type worms are shown. Images are flipped to be in the same direction as the confocal microscopy images in Figure 3. Arrowheads indicate the nucleus. The black lines were drawn to emphasize the boundaries. Scale bar = 20 µm.

    5. Remove the glass slide covering the agarose pad and apply 0.5 µl of the 0.10-µm polystyrene microsphere suspension to the center of the agarose pad (Video 2).

      Video 2. Worm-immobilization


    6. Apply 0.5 µl of 20 mg/ml serotonin hydrochloride/M9 buffer to the polystyrene microsphere suspension on the agarose pad (Figure 1C; Video 2). Serotonin is believed to promote fertilization (Samuel et al., 2001).
      Notes:
      1. Do not mix the polystyrene microspheres and the serotonin in advance because mixing causes the microspheres to aggregate.
      2. Although we usually use serotonin, it is not necessary, and we have observed normal ovulation and fertilization in worms immobilized without serotonin (Takayama and Onami, 2016).
    7. Transfer the worm that is due to ovulate within a few minutes from the 3.5-cm OP50/NGM plate to the drop on the agarose pad by using a worm pick (Video 2).
    8. Arrange the orientation of the worm by using a worm pick such that the oocytes with the injected dye will be situated just beneath the cover glass. Because of the asymmetrical configuration of the anterior and posterior gonads, only one proximal gonad can usually be situated just beneath the cover glass.
    9. Gently place an 18 x 18 mm cover glass on the agarose pad (Video 2).
    10. Stabilize the cover glass by applying four drops of melted VALAP on the four corners of the cover glass by using a P20 micropipette (Figure 1D; Video 2).
    11. Position the sample slide glass on the spinning disk confocal microscope.
    12. Find the worm by using low-magnification objective lenses (e.g., 10x or 20x).
    13. Use a high-magnification water-immersion objective lens to set the field-of-view and the focal plane that best capture the leading sperm in the spermatheca that is expected to fertilize the oocyte.
    14. Start the time-lapse recording when the worm starts ovulating. Typical time-lapse settings for 2D time-lapse imaging are as follows: exposure, 178.6 msec; camera binning, 2; time interval, 0.2 sec (fastest); and duration, 6 min. For 3D time-lapse imaging, the typical settings are as follows: exposure, 50 msec; camera binning, 2; time interval, 1.0 sec; duration, 6 min; z-plane interval, 1 µm; and number of z-planes, 12. The EM-gain is set so that the intensities of the image will be in the predefined range (e.g., 0-4,095 for 12 bit).
    15. Save images as a 16-bit multi-tiff image file.
    16. Capture fluorescence reference images for uneven illumination correction by using a fluorescence reference slide. We typically capture at least 40 different images in the protein film region for the foreground and 5 different images for the background in the 14-bit range (0-16,383).

Data analysis

  1. Installation of plugins for image processing
    1. Download and install ImageJ from https://imagej.nih.gov/ij/download.html.
    2. Download and install mbf-plugins.zip from https://imagej.nih.gov/ij/plugins/mbf/, which contains the BG_Subtraction_from_ROI plugin.
    3. Download, (compile), and install the following plugins from http://so.qbic.riken.jp/cawave/index.html:
      1. Calculator_Plus3.java (Calculator_Plus3.class)
      2. ContourTrack3Stack_.java (ContourTrack3Stack_.class)
      3. Layer_Test2M.java (Layer_Test2M.class, Layer_Test2M$LayeredImageM.class)
      4. ContourTrack4Stack_.java (ContourTrack4Stack_.class)
      5. TrackOocyte_.java (TrackOocyte_.class, LabelHolder.class)
      6. RoughQuantify_.java (RoughQuantify_.class)

  2. Image processing to correct for uneven illumination
    Notes:
    1. The protocol for uneven illumination correction is modified from the original method described by Wolf et al. (2007).
    2. The procedure described below is also presented as a video (Video 3).

      Video 3. Image processing of the reference image to correct for uneven illumination
       

    1. Convert the 16-bit tiff images linearly from 0-16,383 (14-bit range) to 8-bit by using the /Image/Adjust/Brightness/Contrast function and Image/Type/8-bit function.
    2. Average the foreground image (the 40 images of the protein film region in the fluorescence reference slide) by using the Average Intensity projection operation of the Image/Stack/ZProject... function. The resulting file is the averaged foreground image.
    3. Average the background image (the 5 images of the regions outside of the protein film in the fluorescence reference slide) by using the Average Intensity projection operation of the Image/Stack/ZProject... function. The resulting file is the averaged background image.
    4. Subtract the averaged background image from the averaged foreground image by using the Subtract operation of the Calculator_Plus plugin. Open the averaged foreground image and the averaged background image, start the Calculator_Plus plugin, select the averaged foreground image as the i1 and the averaged background image as the i2, select the Subtract operation, and click the OK button. The resulting image file is the reference image.

  3. Image processing for quantification of the time course.
    Notes:
    1. The original data are presented in Takayama and Onami (2016). The example image file can be obtained from the SSBD database (Tohsato et al., 2016)
    2. The entire procedure described below is also presented as five videos (Videos 4-8).
    1. Convert the 16-bit multi-tiff image stack linearly to an 8-bit multi-tiff image stack by using the /Image/Adjust/Brightness/Contrast function and Image/Type/8-bit function. We typically use ranges of 0-2,047 (11 bit), 0-4,095 (12 bit), 0-8,191 (13 bit), or 0-16,383 (14 bit), depending on the intensity range of the image, and convert them to 0-255 (8 bit).
    2. Subtract the background from the ROI by using the BG_subtraction_from_ROI plugin. The background ROI should be a rectangle region that does not include any parts of the worm throughout the time-lapse image stack. We typically set a 60 (w) x 60 (h) pixel region at one corner of the image stack as the ROI and the stdev value as 0. The resulting image stack is the background-corrected image stack.
    3. Normalize the image stack with the reference image by using the Normalize operation of the Calculator_Plus3 plugin. Open the background-corrected image stack and the reference image, start the Calculator_Plus3 plugin, select the background-corrected image stack as the i1 and the reference image as the i2, select Normalize operation, and click the OK button. The resulting image stack is the normalized 8-bit image stack.

      Video 4. Conversion to an 8-bit image stack, background subtraction, and uneven illumination correction (steps C1-C3)


    4. Determine the time slice number of the sperm entry T by visually checking the morphological change in the oocyte tip (Figure 3). The time slice of T-1 is defined as the time 0 slice.
      Note: A sudden protrusion of the oocyte tip is a signature of sperm entry. When performing 3D time-lapse imaging, determine the z slice of sperm entry also.


      Figure 3. Morphological changes in the oocyte tip upon sperm entry. Time-lapse images of the oocyte shape during fertilization in the whole field-of-view (A-D) and in the corresponding magnified region (E-H) indicated by the white box in panel A. The arrowheads in panels C, D, G, and H indicate the sudden protrusion of the oocyte tip upon sperm entry, and hence slice #99 is defined as the time 0 slice. Scale bars in panels A and E = 10 µm.

    5. (Optional) Cut out (1) the time slices earlier than -50 sec and (2) those later than 300 sec because typically they do not have relevant information about fertilization calcium waves.
    6. (Optional) Thin out the time slices to facilitate image processing with an 8-slice interval by using the Deinterleave function. Use the image stack that contains the time 0 slice for subsequent analyses.

      Video 5. Determination of the time 0 slice, cutting slices (earlier than -19.6 sec and later than 250.2 sec in this video), and thinning of the image series (steps C4-C6)


    7. Make a black-white image stack by using the /Image/Adjust/Threshold function. The upper threshold should be set as 255 and the lower threshold to a value that best separates the oocyte region from the background. Use the lower threshold value through the image stack. Check the black background checkbox. The resulting image stack is the black-white image stack.
    8. Remove small debris regions by using the ContourTrack3Stack_ plugin (Figure 4). Open the black-white image stack, start the ContourTrack3Stack_ plugin, and input the upper limit value of the area (pixels) to be removed. A typical input value is 2,000, which means remove a foreground region with an area of less than 2,000 x 0.9 = 1,800. The resulting image stack is the debris-removed black-white image stack.


      Figure 4. The ContourTrack3Stack_ plugin. A. The opened black-white image stack, and the input window of the ContourTrack3Stack_ plugin. B. The resulting image file does not contain debris regions.

    9. Check the shape of the segmented oocyte region by human eye.
      Note: Usually, the oocyte before ovulation cannot be segmented from the other oocytes or the distal gonad regions (Figure 4B).

      Video 6. Conversion to a black-white image stack and removal of debris (steps C7-C9)


    10. Manually correct the segmentation by using the Layer_Test2M plugin (Figure 5). Start the Layer_Test2M plugin, click the Open button, select the normalized 8-bit image stack as the background layer, and select the debris-removed black-white image stack as the foreground layer. Draw or erase by clicking the Erase check box, and manually correct the segmentation by using the pen tool and an interactive pen display. The resulting black-white image stack is the manually corrected black-white image stack.
      Note: The Save button will save the result as a sequence of single-tiff files, whereas the /File/Save as/Tiff... function will save the image stack as a multi-tiff file.


      Figure 5. The Layer_Test2M plugin. A. The window of the Layer_Test2M plugin; B. The layered image stack, in which the semitransparent red foreground image is layered on to the background image.

      Video 7. Manual correction of the segmented region (step C10)


    11. Remove the foreground regions except for the segmented oocyte region by using the ContourTrack4Stack_ plugin (Figure 6). Open the manually corrected black-white image stack, start the ContourTrack4Stack_ plugin, and input the approximate value of the area (pixels) of the oocyte region. A typical input value is 6,000, which means remove foreground regions with areas of less than 6,000 x 2/3 = 4,000 and greater than 6,000 x 4/3 = 8,000.


      Figure 6. The ContourTrack4Stack_ plugin. A. The manually corrected black-white image stack and the input window of the ContourTrack4Stack_ plugin. B. A resulting image stack that does not contain the distal gonad region but does contain other embryonic regions.

    12. (Optional) If the image stack has foreground regions other than the oocyte region, track the oocyte region through the image stack by using the TrackOocyte_ plugin (Figure 7). Start the TrackOocyte_ plugin, and click the oocyte region of interest at the first time slice of the image stack. The resulting image stack is the tracked image stack.


      Figure 7. The TrackOocyte_ plugin. A. By starting the TrackOocyte_ plugin and then clicking the oocyte region (left), the region is tracked through the image stack. B. The resulting image stack of the TrackOocyte_ plugin. Regions other than the clicked region are removed.

    13. If the segmented region has holes (small background regions in the segmented region), fill the holes by using the Process/Binary/Fill_Holes function. The resulting image stack is the black-white mask image stack.
    14. Mask the normalized 8-bit image stack with the black-white mask image stack by using Mask operation of the Calculator_Plus3 plugin. Open the normalized 8-bit image stack and the black-white mask image stack, start the Calculator_Plus3 plugin, and select the normalized 8-bit image stack as the i1 and the black-white mask image stack as the i2, select the Mask operation, and click the OK button. The resulting image stack is the masked 8-bit image stack that contains the segmented oocyte.
    15. Quantify the time course of the calcium fluorescence by using the RoughQuantify_ plugin (Figure 8). Open the masked 8-bit image stack, start the RoughQuantify_ plugin, and input the time 0 slice number and the interval time (sec). The resulting numerical data will consist of the slice number, the mean intensity of the segmented region (F), the time, the ratio (F/F0), the sum of the intensities, the area of the segmented region, the variance, and the standard deviation of the intensities. Save the data as an .xls file for subsequent analyses.


      Figure 8. The RoughQuantify_ plugin. A. The masked 8-bit image stack, and the input window for the time 0 slice number and the time interval. B. The output of the RoughQuantify_ plugin.

      Video 8. Segmentation, tracking, and quantification (steps C11-C15)

Notes

The worm immobilization protocol can be used to image other cellular processes during fertilization such as membrane fusion or cytoskeleton dynamics. For membrane fusion imaging, use the OD58 [oocyte GFP::PH] strain (Audhya et al., 2005) and males of EG4883 [sperm mCherry::histone] (Frøkjaer-Jensen et al., 2008) or the ONA18 [sperm TRP-3::tagRFP-T] strain (Takayama and Onami, 2016) instead of the wild-type N2. For filamentous actin imaging, use the BV67 [lifeact::gfp] strain (Pohl and Bao, 2010).

Recipes

  1. 100 µM Calcium Green-1 dextran solution
    1. Weigh the Calcium Green-1 dextran powder in a 1.5-ml brown tube
    2. Add nuclease-free water to the 1.5-ml brown tube to prepare a 1 mM solution
      Note: Use the dye mol value to calculate the concentration.
    3. Dilute the 1 mM solution to 100 µM with nuclease-free water
    4. Store as 20-µl aliquots in 1.5-ml brown tubes in the dark at -20 °C
  2. Agarose gel pad for microinjection
    1. Prepare 2% or 3.5% agarose/pure water (EMD Millipore, Elix) solution in a 50-ml screw-cap bottle
    2. Make agarose pads on 24 x 55 mm cover glasses
    Note: We use Agarose-LE, Classic Type (Nacalai Tesque) or Agarose (Thermo Fisher Scientific, Invitrogen) for the 2% solution or 3.5% solution, respectively.
  3. Sterilized 1 M MgSO4 solution
    1. Dissolve 123.24 g MgSO4·7H2O in 500 ml of pure (Elix) water
    2. Autoclave at 121 °C for 20 min
    3. Store at room temperature
  4. M9 buffer
    1. Dissolve the following in 1 L of pure (Elix) water
      3 g KH2PO4
      6 g Na2HPO4
      5 g NaCl
    2. Autoclave at 121 °C for 20 min, then add 1 ml of sterilized 1 M MgSO4 solution
    3. Store at room temperature
  5. 10% NuSieve GTG agarose pad
    1. Mix well 1.0 g of NuSieve GTG agarose with 10 ml of M9 buffer in a 50-ml screw-cap glass bottle by using a micro spatula
    2. Melt the NuSieve GTG agarose/M9 buffer mixture by using a microwave oven
    3. Cool the 10% NuSieve GTG agarose/M9 buffer mix at room temperature to allow it to solidify
    4. Store at room temperature
  6. VALAP
    1. Mix Vaseline, lanolin, and paraffin at a 2:2:1 (weight) ratio in a 100-ml beaker
    2. Cover the beaker with aluminum foil
    3. Melt the contents of the beaker at 130 °C on a hot plate
    4. Store at room temperature
  7. Injection recovery solution
    1. Dissolve 2 g of glucose in 50 ml of M9 buffer
    2. Sterilize by membrane filtration
    3. Prepare 500-µl aliquots in 1.5-ml tubes
    4. Add 5 µl of 2.0% gelatin solution to the 500-µl aliquot when needed
      Note: Gelatin is added to prevent the worm from sticking to the tip.
    5. Store at room temperature
  8. 20 mg/ml serotonin hydrochloride/M9 buffer
    1. Weigh the serotonin hydrochloride in a 1.5-ml tube
    2. Add M9 buffer to the 1.5-ml tube to prepare a 20 mg/ml solution
    3. Prepare 10-µl aliquots in 1.5-ml tubes
    4. Store in a sealed bag at -20 °C
    Note: Storing in a sealed bag prevents the solution from acquiring a brownish color.
  9. Fluorescence reference slide
    1. Draw a circle (approximately 5 mm in diameter) on an 18 x 18 mm cover glass by using a diamond pen to indicate the region of the fluorescent protein film. The side with the circle is the upper side of the cover glass
    2. Apply 10 µl of 0.2% BSA-fluorescein solution to the circle on the upper side of the cover glass
    3. Incubate the cover glass in a dark humidified chamber for 30 min at room temperature
    4. Dip the cover glass into approximately 40 ml of pure (Elix) water in a 50-ml beaker to wash it. Repeat this washing step with another approximately 40 ml of water
    5. Carefully remove the water drops with a lint-free wipe (Kimwipes)
      Note: Do not touch the protein film region with the wipe.
    6. Leave the cover glass to dry at room temperature
    7. Flip and place the cover glass on a glass slide so that the upper side of the cover glass faces the glass slide
    8. Seal the edges of the cover glass with transparent (Scotch) tape
    9. Store at -20 °C in the dark

Acknowledgments

The protocol is used in Takayama and Onami (2016). The protocol for the microinjection of a chemical calcium indicator dye is based on that described by Samuel et al. (2001). The immobilization method is based on the method of Kim et al. (2013). The uneven illumination correction is derived from the work of Wolf et al. (2007). The Calculator_Plus3 plugin was modified from the Calculator_Plus plugin, which was developed by Wayne Rasband and Gabriel Landini. Portions of the ContourTrack3Stack_, ContourTrack4Stack_, and TrackOocyte_ plugin codes were reused from a program written by Takuya Maeda. We thank Asako Sugimoto for her advice regarding the choice of microscopy filters. We also thank Koji Kyoda and Rie Furushima for their assistance. This work was supported, in part, by the National Bioscience Database Center (NBDC) of the Japan Science and Technology Agency (S.O.), by the Strategic Programs for R&D (President’s Discretionary Fund) of RIKEN (S.O.), and by JSPS KAKENHI Grant Number 15K18547 (J.T.). The authors declare that they have no competing interests.

References

  1. Audhya, A., Hyndman, F., McLeod, I. X., Maddox, A. S., Yates, J. R., 3rd, Desai, A. and Oegema, K. (2005). A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans. J Cell Biol 171(2): 267-279.
  2. Evans, T. C. (2006). Transformation and microinjection. In: WormBook (Ed.). The C. elegans Research Community. WormBook.
  3. Frøkjaer-Jensen, C., Davis, M. W., Hopkins, C. E., Newman, B. J., Thummel, J. M., Olesen, S. P., Grunnet, M. and Jorgensen, E. M. (2008). Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 40(11): 1375-1383.
  4. Kim, E., Sun, L., Gabel, C. V. and Fang-Yen, C. (2013). Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization. PLoS One 8(1): e53419.
  5. Pohl, C. and Bao, Z. (2010). Chiral forces organize left-right patterning in C. elegans by uncoupling midline and anteroposterior axis. Dev Cell 19(3): 402-412.
  6. Samuel, A. D., Murthy, V. N. and Hengartner, M. O. (2001). Calcium dynamics during fertilization in C. elegans. BMC Dev Biol 1: 8.
  7. Takayama, J. and Onami, S. (2016). The sperm TRP-3 channel mediates the onset of a Ca2+ wave in the fertilized C. elegans oocyte. Cell Rep 15(3): 625-637.
  8. Tohsato, Y., Ho, K. H., Kyoda, K. and Onami, S. (2016). SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena. Bioinformatics 32(22): 3471-3479.
  9. Wolf, D. E., Samarasekera, C. and Swedlow, J. R. (2007). Quantitative analysis of digital microscope images. Methods Cell Biol 81: 365-396.

简介

受精钙波是动物发育的保守触发因素; 然而,这些波的遗传分析由于在体内成像难以受精而受到限制。 在这里,我们描述了在遗传动物模型秀丽隐杆线虫中的体内受精期间成像钙动力学的方案。 该方案包括化学钙指示剂的种系显微注射,蠕虫固定,活体成像和图像处理,其量化在排卵期间在视野内移动的卵母细胞区域中的钙荧光。 该成像方案也可以用于在体内受精期间对其他细胞过程进行成像。 线虫,如膜融合和细胞骨架动力学。

受精钙波在卵活化中起关键作用,并通过使用体外施肥系统在各种生物体中进行了分析。 线虫秀丽隐杆线虫由于其半透明的身体,适合于在体内成像受精。 受精钙成像在C. 塞缪尔等人报道了使用化学钙指示剂的线虫。 (2001)。 我们在这里描述了通过应用高速旋转盘共焦显微镜和图像处理方法从成像方法修改的方案,其在排卵期间分割在视场中移动的受精卵母细胞区域。 该协议能够精确定量描述钙波的时间动力学和波形图的遗传分析。

关键字:受精, 受精钙波, 钙波, 秀丽隐杆线虫, 实时成像, 钙成像, 图像处理

材料和试剂

  1. 玻璃毛细丝(NARISHIGE,目录号:GDC-1)
  2. 24 x 55毫米封面玻璃(松本玻璃,目录号:C024551)
  3. 76 x 26毫米玻璃片(松本玻璃,目录号:S011110)
  4. 18×18毫米玻璃盖(厚度0.16-0.19毫米; 1S/1.5号)(松本玻璃,特殊订购产品)
  5. 微铲(例如,,AS ONE,目录号:6-524-01)
  6. 1.5ml棕色管(例如,FUKAEKASEI,WATSON,目录号:131-915BR)
  7. 铝箔(例如UACJ箔公司)
  8. 注射器驱动膜过滤器(EMD Millipore,目录号:SLGS033SS)
  9. 19(W)×0.18(T)mm乙烯胶带(3M,目录号:35-WHITE-3/4)
  10. 无绒擦拭(KCWW,Kimwipes,目录号:S-200)
  11. 暗室(例如,,Ina-optika,型号:FB-35BL)
    注意:我们在内盖上形成大约7×10.5厘米的矩形孔。
  12. N2野生型C。 elegans
  13. 用于显微注射的琼脂糖凝胶垫
    1. Agarose-LE,经典型(Nacalai Tesque,目录号:01157-66)
    2. 或琼脂糖(Thermo Fisher Scientific,Invitrogen,目录号:15510-027)
      注意:本产品已停产。
  14. 液体石蜡(Wako Pure Chemical Industries,目录号:164-00476)
  15. 0.10微米聚苯乙烯微球悬浮液(Polysciences,目录号:00876-15)
    注意:

    1. 在1.5 ml管中准备大约500μl等分试样
    2. 存储在4°C
  16. 盐酸5-羟色胺(Sigma-Aldrich,目录号:H9523)
  17. 钙绿1葡聚糖,钾盐,10,000MW阴离子(Thermo Fisher Scientific,Molecular Probes ,目录号:C3713)
  18. 无核酸酶水(Promega,目录号:P1193)
  19. 磷酸二氢钾(KH 2 PO 4)(和光纯药,目录号:169-04245)
  20. 磷酸氢二钠(Na 2 HPO 4)(和光纯药,目录号:197-02865)
  21. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  22. 硫酸镁七水合物(MgSO 4·7H 2 O)(和光纯药,目录号:131-00405)
  23. NuSieve GTG琼脂糖(Lonza,目录号:50080)
  24. 凡士林(白凡士林)(Kozakai Pharmaceutical)
  25. 羊毛脂(Sigma-Aldrich,目录号:L7387)
  26. 石蜡颗粒(例如,Seiwa)
  27. D(+) - 葡萄糖(Wako Pure Chemical Industries,目录号:041-00595)
    注意:本产品已停产。
  28. 0.2%BSA-荧光素溶液(溶于不含核酸酶的水中)
    注意:使用前请先准备好。
  29. 2.0%明胶溶液
  30. BSA-荧光素(Thermo Fisher Scientific,Molecular Probes TM,目录号:A23015)
  31. 100μM钙绿葡聚糖溶液(参见食谱)
  32. 灭菌的1M MgSO 4溶液(参见食谱)
  33. M9缓冲区(见配方)
  34. 10%NuSieve GTG琼脂糖/M9垫(见食谱)
  35. VALAP(见配方)
  36. 注射液回收溶液(参见食谱)
  37. 20 mg/ml盐酸羟色胺/M9缓冲液(见配方)
  38. 荧光参考幻灯片(见配方)

设备

  1. 拔针器(NARISHIGE,型号:PC-10)
  2. 微量注射器(Eppendorf,型号:FemtoJet Microinjector)
  3. 具有操纵器的倒置显微镜(例如,Leica Microsystems,型号:DMIRB; NARISHIGE型号:MMO-220A,MO-202U,MN-4)
  4. 热板(例如,IKA,目录号:C-MAG HP7)
  5. 微波炉(例如,松下,型号:NE-EH212)
    注意:本产品已停产。
  6. 显微镜(例如,Leica Microsystems,型号:DMRE; Nikon Instruments,型号:Ti-E)
  7. 50毫升螺旋盖玻璃瓶(例如,AS ONE,目录号:1-4568-01)
  8. 100ml烧杯(例如,ASAHI Glass,AGC,目录号:1000BK100)
  9. 50ml烧杯(例如,ASAHI Glass,AGC,目录号:1000BK50)
  10. 钻石笔(例如,,Ogura Jewel Industry,型号:D点笔)
  11. 高倍率水浸物镜(例如,Leica Microsystems,型号:HCX PL Apo 63x/1.20W corr; Nikon Instruments,型号:Plan Apo VC 60XA/1.20W)
  12. 旋转盘共焦单元(横河电机,型号:CSU-X1)
  13. EM-CCD摄像头(Andor Technology,型号:iXon DU-897)
  14. Ar/Kr激光(IDEX,Melles Griot,型号:643-YB-A01)
  15. 过滤器/快门控制器(Ludl Electronic Products,型号:Mac 6000,目录号:73006081,73006042,73006001)
  16. 488nm激光线滤波器(IDEX,Semrock,目录号:LL01-488)
  17. 分色镜(IDEX,Semrock,目录号:Di01-T488)
  18. 长通屏障滤波器(IDEX,Semrock,目录号:BLP01-488R)
  19. 用于图像采集的Windows 7计算机
  20. 用于图像处理的Mac计算机(例如,,10.6.8或更高版本)
  21. 互动笔显示(Wacom,型号:Cintiq 21UX)

软件

  1. ImageJ(版本1.49v或更新版本)( https://imagej。 nih.gov/ij/download.html
  2. 图像采集软件(例如,,Andor Technology,iQ [ver.1.10.3或更高版本]或iQ2 [ver.2.9.1或更高版本])

程序

注意:该协议是从Samuel等人描述的原始方法进行修改的。 (2001)。

  1. 显微注射化学钙指示剂染料
    1. 通过使用标准方法将100μM钙绿葡萄糖溶液注入妊娠成年雌雄同体前生殖系的远端臂。 elegans DNA显微注射(参见Evans,2006)。我们使用GDC-1玻璃毛细管,双拉模式下由PC-10拉拔器拉出的细丝。此外,我们在24 x 55 mm的玻璃上使用2%或3.5%琼脂糖凝胶垫进行蠕虫固定和液体石蜡,以保护样品免受干燥。
      注意:
      1. 如Samuel等人所述,填满大约一半的性腺(2001)。注射过多的染料经常会导致恢复后的性腺异常异常。
      2. 我们总是每个琼脂糖垫只放置一个蠕虫进行注射,以最大限度地减少蠕虫固定的时间,从而最大限度地减少损伤。
      3. 我们通常在一组实验(例如一种基因型)中注射四至六个蠕虫,并在一天内进行两组实验,间隔为2小时。大约一半的注射蠕虫可以对每组实验进行成像。
    2. 要恢复注射的蠕虫,使用P20微量移液管将大约1μl的注射液回收溶液涂抹在蠕虫上
    3. 通过使用P20微量吸管,将注射的蠕虫吸入注射液回收溶液中,并将其从琼脂糖垫转移到3.5厘米的OP50/NGM板上。
      注意:从采摘到转移的单次注射程序(步骤A1-A3)应在约5分钟内完成。
    4. 允许注射的蠕虫在室内的黑暗室中恢复4-6小时。
    5. 使用立体荧光显微镜检查注射的染料是否并入蠕虫的卵母细胞 注意:Sloppy注射可能导致染料渗入细胞中(Samuel et al。,2001)。

  2. 蠕虫准备和成像
    注意:蠕虫固定方法从Kim等人描述的原始方法进行了修改。 (2013)。
    1. 熔化VALAP在130°C的热板上。
    2. 准备10%琼脂糖凝胶垫。
      1. 准备两个双层玻片(图1A)。该图像中的磁带堆叠厚度为0.36 mm
      2. 在微波炉中将10%NuSieve GTG琼脂糖/M9缓冲液熔化。
      3. 通过使用微型刮刀将一滴熔化的10%NuSieve GTG琼脂糖/M9缓冲液施加到两个胶带玻片之间的载玻片上(图1A;视频1)。
      4. 用另一张玻璃片盖住熔化的琼脂糖滴(图1B;视频1)。
      5. 在室温下孵育至少30分钟以固化琼脂糖垫。保持玻璃片覆盖的琼脂糖垫直到使用。


        图1.样品制备A.将一滴10%琼脂糖通过使用微型刮刀施加到两个双层隔片之间的载玻片上。 B.将载玻片放在10%琼脂糖滴上。 C.将一滴微球悬浮液和一滴血清素/M9溶液施加到立体显微镜台上的琼脂糖垫上。 D.准备成像的样品,其中玻璃板上的VALAP在琼脂糖垫上稳定(未注射的蠕虫固定在该图像中)。

        Video 1. Preparation of a 10% agarose gel pad

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player



    3. 允许聚苯乙烯微球悬浮液和盐酸5-羟色胺/M9缓冲液在使用前在室温下保温至少30分钟。
    4. 检查注射的蠕虫的卵母细胞的形状,使用立体荧光显微镜确定排卵的时间。前卵母细胞的形状应呈矩形,其核应位于精子细胞远端的皮层区域(图2)。


      图2.基于卵母细胞形状推断排卵时间。A.在几分钟内出现准备排卵的卵母细胞。卵母细胞形状是矩形的,面向下一个卵母细胞的边界看起来像一条直线(由黑线表示),并且细胞核靠近精子细胞远端的皮层区域。 B.卵母细胞未准备成像。边界具有弧形外观(由黑线表示),细胞核不靠近远端皮层。显示非注射野生型蠕虫的差分干涉对比图像。图像被翻转成与图3中的共聚焦显微镜图像相同的方向。箭头指示细胞核。绘制黑线以强调边界。比例尺=20μm。

    5. 取出覆盖琼脂糖垫的玻片,将0.5微升的0.10微米聚苯乙烯微球悬浮液加到琼脂糖垫的中心(视频2)。

      Video 2. Worm-immobilization

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player



    6. 在琼脂糖垫上的聚苯乙烯微球悬浮液中加入0.5μl20mg/ml的羟色胺盐酸盐/M9缓冲液(图1C; Video 2)。据信,血清素促进受精(Samuel等人,2001)。
      注意:
      1. 不要预先混合聚苯乙烯微球和5-羟色胺,因为混合导致微球聚集。
      2. 虽然我们通常使用5-羟色胺,但并不是必需的,我们已经观察到没有血清素固定的蠕虫中的正常排卵和受精(Takayama和Onami,2016)。
    7. 通过使用蠕虫选择(视频2),将距离3.5厘米OP50/NGM板几分钟内排卵的蠕虫转移到琼脂糖垫上的下降。
    8. 通过使用蠕虫选择来排列蠕虫的方向,使得具有注射的染料的卵母细胞将位于盖玻片的正下方。由于前和后性腺的不对称构型,通常只有一个近端性腺位于护罩玻璃下方。
    9. 轻轻地将18 x 18毫米的盖玻片放在琼脂糖垫上(视频2)。
    10. 通过使用P20微量移液器(图1D;视频2),通过在玻璃杯的四个角落涂上四滴熔化的VALAP来稳定玻璃罩。
    11. 将样品载玻片放在旋转盘共聚焦显微镜上
    12. 通过使用低倍率物镜(例如,10x或20x)来查找蠕虫。
    13. 使用高放大倍率的浸水式物镜设置最佳捕获精母细胞中预期能够对卵母细胞施肥的主要精子的视场和焦平面。
    14. 当蠕虫开始排卵时开始延时记录。 2D延时成像的典型延时设置如下:曝光178.6毫秒;相机合并,2;时间间隔0.2秒(最快);和持续时间,6分钟。对于3D延时成像,典型设置如下:曝光50毫秒;相机合并,2;时间间隔1.0秒;持续时间6分钟; z平面间隔,1μm;和z平面的数量12.设置EM增益使得图像的强度将在预定义的范围内(例如,对于12位,为0-4,095)。 >
    15. 将图像保存为16位多图像文件。
    16. 通过使用荧光参考幻灯片捕获不均匀照明校正的荧光参考图像。我们通常在前景的蛋白质胶片区域捕获至少40个不同的图像,在14位范围内捕获5种不同的图像(0-16,383)。

数据分析

  1. 安装用于图像处理的插件
    1. https://imagej.nih.gov/ij下载并安装ImageJ /download.html
    2. https://imagej。下载并安装mbf-plugins.zip。 nih.gov/ij/plugins/mbf/,其中包含BG_Subtraction_from_ROI插件。
    3. 下载(编译)并从 http: //so.qbic.riken.jp/cawave/index.html
      1. Calculator_Plus3.java(Calculator_Plus3.class)
      2. ContourTrack3Stack_.java(ContourTrack3Stack_.class)
      3. Layer_Test2M.java(Layer_Test2M.class,Layer_Test2M $ LayeredImageM.class)
      4. ContourTrack4Stack_.java(ContourTrack4Stack_.class)
      5. TrackOocyte_.java(TrackOocyte_.class,LabelHolder.class)
      6. RoughQuantify_.java(RoughQuantify_.class)

  2. 图像处理以校正不均匀照明
    注意:
    1. 用于不均匀照明校正的协议由Wolf等人描述的原始方法进行修改。 (2007)。
    2. 下面描述的过程也被视为视频(视频3)。

      Video 3. Image processing of the reference image to correct for uneven illumination

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

       

    1. 使用/Image/Adjust/Brightness/Contrast功能和Image/Type/8-bit功能,将16位tiff图像从0-16,383(14位范围)线性转换为8位。
    2. 通过使用Image/Stack/ZProject ...功能的平均强度投影操作,平均前景图像(荧光参考幻灯片中蛋白质膜区域的40张图像)。生成的文件是平均的前景图像。
    3. 通过使用Image/Stack/ZProject ...功能的平均强度投影操作来平均背景图像(荧光参考幻灯片中的蛋白质膜外部的5个图像)。生成的文件是平均的背景图像。
    4. 通过使用Calculator_Plus插件的减法操作,从平均的前景图像中减去平均的背景图像。打开平均的前景图像和平均的背景图像,启动Calculator_Plus插件,选择平均的前景图像为i1,平均的背景图像为i2,选择"减法",然后单击"确定"按钮。生成的图像文件是参考图像。

  3. 用于量化时间过程的图像处理。
    注意:
    1. 原始数据呈现于高山和Onami(2016)。示例图像文件可以从 SSBD数据库(Tohsato et al。,2016)
    2. 下面描述的整个过程也被呈现为五个视频(视频4-8)。
    1. 通过使用/Image/Adjust/Brightness/Contrast功能和Image/Type/8-bit功能,将16位多Tiff图像堆栈线性转换为8位多图形图像堆栈。取决于图像的强度范围,我们通常使用0-2,047(11位),0-4,095(12位),0-8,191(13位)或0-16,383(14位)的范围,并将其转换到0-255(8位)。
      Video 4. Conversion to an 8-bit image stack, background subtraction, and uneven illumination correction (steps C1-C3)

      To play the video, you need to install a newer version of Adobe Flash Player.

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    2. 通过目视检查卵母细胞尖端的形态变化来确定精子入口的时间片数(图3)。时间片T-1 定义为时间0片。
      注意:卵母细胞突然突出是精子进入的标志。执行3D延时成像时,也可以确定精子输入的z切片。


      图3.精子进入时卵母细胞尖端的形态学变化在整个视野(AD)和相应的放大区域(EH)中受精期间卵母细胞形状的延时图像由图A中的白盒指示。图C,D,G和H中的箭头表示在精子进入时卵母细胞尖端突然突出,因此切片#99被定义为时间0切片。面板A和E中的比例尺为10μm。

    3. (可选)切出(1)-50秒之前的时间片段,(2)超过300秒的时间片段,因为它们没有关于受精钙波的相关信息。
    4. (可选)通过使用Deinterleave功能,缩短时间片以便于以8片段间隔进行图像处理。使用包含时间0片的图像堆栈进行后续分析。

      Video 5. Determination of the time 0 slice, cutting slices (earlier than -19.6 sec and later than 250.2 sec in this video), and thinning of the image series (steps C4-C6)

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    5. 使用/Image/Adjust/Threshold功能进行黑白图像叠加。应将上限阈值设置为255,将下限阈值设置为最好将卵母细胞区域与背景区分开的值。通过图像堆栈使用较低的阈值。检查黑色背景复选框。所产生的图像堆栈是黑白图像堆栈。
    6. 使用ContourTrack3Stack_插件去除小碎片区域(图4)。打开黑白图像堆栈,启动ContourTrack3Stack_插件,并输入要删除的区域(像素)的上限值。典型的输入值为2,000,这意味着删除面积小于2,000 x 0.9 = 1,800的前景区域。所得到的图像堆栈是碎片去除的黑白图像堆栈。


      图4. ContourTrack3Stack_插件。 A.打开的黑白图像堆栈以及ContourTrack3Stack_插件的输入窗口。 B.生成的图像文件不包含碎片区域。

    7. 用人眼检查分段卵母细胞区域的形状。
      注意:通常,排卵前的卵母细胞不能从其他卵母细胞或远端性腺分裂(图4B)。

      Video 6. Conversion to a black-white image stack and removal of debris (steps C7-C9)

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    8. 使用Layer_Test2M插件手动更正分割(图5)。启动Layer_Test2M插件,单击打开按钮,选择标准化的8位图像堆栈作为背景层,并选择碎片去除的黑白图像堆栈作为前景层。通过单击删除复选框绘制或删除,并使用笔工具和交互式笔显示来手动更正分割。所产生的黑白图像堆栈是手动校正的黑白图像堆栈。
      注意:"保存"按钮将会将结果保存为单个tiff文件的顺序,而/File/Save as/Tiff ...功能会将映像堆栈保存为多tiff文件。


      图5. Layer_Test2M插件。 A. Layer_Test2M插件的窗口; B.分层图像堆栈,其中半透明红色前景图像分层到背景图像上。

      Video 7. Manual correction of the segmented region (step C10)

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    9. 通过使用ContourTrack4Stack_插件,除去分割的卵母细胞区域之外的前景区域(图6)。打开手动校正的黑白图像堆栈,启动ContourTrack4Stack_插件,并输入卵母细胞区域(像素)的近似值。典型的输入值为6000,这意味着去除区域小于6,000 x 2/3 = 4,000且大于6,000 x 4/3 = 8,000的前景区域。


      图6. ContourTrack4Stack_插件。 A.手动校正的黑白图像堆栈和ContourTrack4Stack_插件的输入窗口。 B.一个不包含远端性腺区域,但包含其他胚胎区域的图像堆栈。

    10. (可选)如果图像堆栈具有卵母细胞区域以外的前景区域,请使用TrackOocyte_插件通过图像堆栈跟踪卵母细胞区域(图7)。启动TrackOocyte_插件,然后在图像堆栈的第一个时间片段单击感兴趣的卵母细胞区域。所得到的图像堆栈是跟踪的图像堆栈。


      图7. TrackOocyte_插件。 A.通过启动TrackOocyte_插件,然后单击卵母细胞区域(左),该区域将通过图像堆栈进行跟踪。 B.产生的TrackOocyte_插件的图像堆栈。除了点击区域以外的区域被删除。

    11. 如果分段区域具有孔(分段区域中的小背景区域),则使用Process/Binary/Fill_Holes函数填充孔。所得到的图像堆栈是黑白掩模图像堆栈。
    12. 通过使用Calculator_Plus3插件的Mask操作,屏蔽黑白掩模图像堆栈的归一化8位图像堆栈。打开标准化的8位图像堆栈和黑白掩模图像堆栈,启动Calculator_Plus3插件,并选择归一化的8位图像堆栈作为i1,黑白掩模图像堆栈作为i2,选择"掩码" ,然后单击确定按钮。所产生的图像堆栈是包含分段卵母细胞的被屏蔽的8位图像堆栈。
    13. 使用RoughQuantify_插件量化钙荧光的时间过程(图8)。打开屏蔽的8位映像堆栈,启动RoughQuantify_插件,并输入时间0片数和间隔时间(秒)。所得到的数值数据将由切片数,分段区域的平均强度(F),时间,比值(F/F 0 ),强度之和,面积分段区域,方差和强度的标准偏差。将数据保存为.xls文件以供后续分析。


      图8.RoughQuantify_插件。 A.屏蔽的8位映像堆栈,以及时间0片段编号和时间间隔的输入窗口。 B. RoughQuantify_插件的输出。

      Video 8. Segmentation, tracking, and quantification (steps C11-C15)

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笔记

蠕虫固定方案可以用于在受精期间的其他细胞过程,如膜融合或细胞骨架动力学。对于膜融合成像,使用OD58 [卵母细胞GFP :: PH]菌株(Audhya等人,2005)和EG4883雄性[精子mCherry ::组蛋白](Frøkjaer-Jensen等) (2008))或ONA18 [精子TRP-3 :: tagRFP-T]菌株(Takayama和Onami,2016),而不是野生型N 2。对于丝状肌动蛋白成像,使用BV67 [lifeact :: gfp]菌株(Pohl和Bao,2010)。

食谱

  1. 100μM钙绿葡聚糖溶液
    1. 在1.5 ml棕色管中称量"Green-1"葡聚糖粉末
    2. 将无核酸酶的水加入到1.5-ml棕色管中以制备1mM溶液 注意:使用染料摩尔值计算浓度。
    3. 用无核酸酶的水将1mM溶液稀释至100μM
    4. 在-20°C的黑暗中储存为20-μl等分试样放入1.5-ml棕色管中
  2. 用于显微注射的琼脂糖凝胶垫
    1. 准备2%或3.5%琼脂糖/纯水(EMD Millipore,Elix)溶液在50ml螺旋盖瓶中
    2. 在24 x 55毫米的护目镜上制作琼脂垫
    注意:我们分别使用Agarose-LE,经典型(Nacalai Tesque)或琼脂糖(Thermo Fisher Scientific,Invitrogen)作为2%溶液或3.5%溶液。
  3. 灭菌的1M MgSO 4溶液
    1. 将123.24g MgSO 4·7H 2 O溶解在500ml纯(Elix)水中
    2. 在121℃高压灭菌20分钟
    3. 在室温下存放
  4. M9缓冲液
    1. 在1升纯(Elix)水中溶解以下物质 3g KH 2 PO 4
      6g Na 2 HPO 4
      5克NaCl
    2. 在121℃高压灭菌20分钟,然后加入1毫升灭菌的1M MgSO 4溶液
    3. 在室温下存放
  5. 10%NuSieve GTG琼脂糖垫
    1. 通过使用微量铲将1.0g NuSieve GTG琼脂糖与10ml M9缓冲液在50ml螺旋盖玻璃瓶中混合,
    2. 通过使用微波炉将NuSieve GTG琼脂糖/M9缓冲液混合物熔化
    3. 在室温下冷却10%NuSieve GTG琼脂糖/M9缓冲液混合物,使其固化
    4. 在室温下存放
  6. VALAP
    1. 将凡士林,羊毛脂和石蜡以2:2:1(重量)的比例混合在100 ml烧杯中,
    2. 用铝箔盖住烧杯
    3. 将130℃的烧杯内容物熔化在热板上
    4. 在室温下存放
  7. 注射液回收溶液
    1. 将2 g葡萄糖溶于50ml M9缓冲液中
    2. 通过膜过滤灭菌
    3. 在1.5 ml管中准备500μl等分试样
    4. 当需要时,将5μl的2.0%明胶溶液加入到500μl等分试样中 注意:添加明胶以防止蠕虫粘到尖端。
    5. 在室温下存放
  8. 20mg/ml盐酸羟色胺/M9缓冲液
    1. 称量盐酸5-羟色胺在一个1.5毫升的管子
    2. 将M9缓冲液加入到1.5 ml管中以制备20 mg/ml溶液
    3. 准备10μl等分试样在1.5 ml管中
    4. 存放在-20°C的密封袋中
    注意:存放在密封袋中可防止溶液获得褐色。
  9. 荧光参考幻灯片
    1. 通过使用金刚石笔来表示荧光蛋白膜的区域,在18×18mm的盖玻璃上绘制一个圆圈(直径约5毫米)。圆的一侧是玻璃的上侧
    2. 将10μl0.2%BSA-荧光素溶液涂抹在玻璃罩上侧的圆上
    3. 在深色加湿室中孵育玻璃盖,室温下放置30分钟
    4. 将盖玻璃浸入50ml烧杯中的约40ml纯(Elix)水中以进行洗涤。用另一个大约40毫升的水重复这个洗涤步骤
    5. 用无毛巾擦拭水滴(Kimwipes)
      注意:不要用擦拭物触摸蛋白质膜区域。
    6. 将盖玻片在室温下干燥
    7. 翻转并将盖玻片放在玻璃滑块上,以使盖玻璃的上侧面向玻璃片
    8. 用透明(苏格兰)胶带
      密封盖玻璃的边缘
    9. 储存于-20°C,黑暗中

致谢

该协议用于高山和Onami(2016)。用于显微注射化学钙指示剂染料的方案基于Samuel等人描述的方案。 (2001)。固定方法基于Kim等人的方法。 (2013)。不均匀的照明校正来自Wolf等人的工作。 (2007)。 Calculator_Plus3插件是由由Wayne Rasband和Gabriel Landini开发的Calculator_Plus插件修改的。 ContourTrack3Stack_,ContourTrack4Stack_和TrackOocyte_插件代码的部分由Takuya Maeda编写的程序重复使用。感谢Asako Sugimoto关于选择显微镜过滤器的建议。我们还感谢Kyji Kyoda和Rie Furushima的协助。这项工作部分得到了日本科学技术厅(SO)的国家生物科学数据库中心(NBDC),RIKEN(SO)的研发(总统自选资金)战略计划和JSPS的支持KAKENHI Grant Number 15K18547(JT)。作者宣称他们没有竞争的利益。

参考文献

  1. Audhya,A.,Hyndman,F.,McLeod,IX,Maddox,AS,Yates,JR,3rd,Desai,A。和Oegema,K。(2005)。含有Sm蛋白CAR-1和RNA解旋酶CGH-1的复合物是胚胎细胞分裂所必需的,秀丽隐杆线虫。 J Cell Biol 171(2):267-279。
  2. Evans,TC(2006)。转化和显微注射。/a> In:WormBook(Ed。)。 C。线虫研究社区。 WormBook 。
  3. Frøkjaer-Jensen,C.,Davis,MW,Hopkins,CE,Newman,BJ,Thummel,JM,Olesen,SP,Grunnet,M.and Jorgensen,EM(2008)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/18953339"target ="_ blank">在秀丽隐杆线虫中单拷贝插入转基因 Nat Genet 40(11):1375-1383。
  4. Kim,E.,Sun,L.,Gabel,CV and Fang-Yen,C。(2013)。  使用纳米颗粒介导的固定化的秀丽隐杆线虫的长期成像 8(1): e53419。
  5. Pohl,C.and Bao,Z.(2010)。  通过解开中线和前后轴。 Dev Cell 19(3):402-412。
  6. Samuel,AD,Murthy,VN和Hengartner,MO(2001)。  钙受精过程中的钙动力学。 elegans BMC Dev Biol 1:8.
  7. 高山,J.和Onami,S。(2016)。精子TRP-3通道介导受精C中的Ca 2 + 波的发生。电针卵母细胞。 Cell Rep 15(3):625-637。
  8. Tohsato,Y.,Ho,KH,Kyoda,K.and Onami,S。(2016)。 SSBD:生物现象时空动态定量数据的数据库。生物信息学32(22):3471-3479。
  9. Wolf,DE,Samarasekera,C.和Swedlow,JR(2007)。数字显微镜图像的定量分析。方法细胞周期81:365-396。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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