Analysis of Ca2+ transients

Analysis of ciliary Ca2+ transient in mouse embryonic node

Katsutoshi Mizuno^1,2

1. RIKEN Center for Biosystems Dynamics Research

2. University of Fukui, School for Medical Sciences

From “Role of Ca²⁺ transients at the node of the mouse embryo in breaking of left-right symmetry”, DOI: 10.1126/sciadv.aba1195

Ca²⁺ imaging

1. Recover E7.5 mouse embryo at E7.5 in HEPES-buffered DMEM (pH 7.2) supplemented with 10% fetal bovine serum. For an experiment, only embryos around early head fold stage-1, 2 somites stage embryos can be used.

2. Maintain embryos with roller culture, in DMEM supplemented with 75% rat serum (pH 7.2) until use.

3. Excise a distal portion of each embryo including the node with a needle.

4. Place the embryo in a chamber made of a glass-slide with a silicone rubber spacer (200 µm thick) and cover with a coverslip. Mounting solution should be DMEM supplemented with 75% rat serum.

5. Place the slide on a microscope and incubate the embryo for 10 min at 37°C, 5% CO2 before the observation.

6. For ciliary GCaMP6 and cytoplasmic RGECO1, we used Olympus IX83 equipped with the CSU-W1 confocal unit (Yokogawa) with 488 nm and 561 nm laser, and with UPLSAPO 60XW or UPLSAPO 40XS objective (Olympus).

7. Images were recorded simultaneously with two synchronized EMCCD cameras (iXon Ultra 888, Andor). Exposure time was set to 200 ms, and XYT images were obtained at a rate of 4 frames per second. The duration of observation was ~2 min, and movies were obtained from several depths within the range of 15 to 25 µm from the base of the node. If necessary, XYZT images were obtained with a piezo actuator (Physik Instrumente), with a Z depth of 1.5 µm and with seven or eight sections.

Analysis of ciliary Ca²⁺ transients

In our analysis, I generally use Fiji/ImageJ. In our case, we use GCaMP6 image, which is Ca2+ sensor, and mCherry image, which is used as a reference of ciliary position. To set a region of interest (ROI) for each cilium, which is highly mobile, we make a binary image utilizing the mCherry signal.

1. Open an image (both mCherry and GCaMP6) for analysis with Fiji. Measure background signal outside of cilia, and subtract from the original images.

2. Duplicate mCherry image. This duplicated image is used to create the “Mask” image to extract only the ciliary region. Adjust threshold so that only cilia can be visible and make a binary image (“Image/Adjust/Threshold...”). Before binarize, filter with Gaussian filter, or other filters can be effective to extract cilia region without any noise. Push “Apply” button and execute binarization. Name the image as “binary”. Now the image has 0 and 255 values.

3. Using “Math” tool, divide the image by 255. Now, image has 0 and 1 pixel values (“0/1 image”).

4. Next, we want a mask image with 1 and “NA” for the background to extract the image. For this purpose, convert the “0/1” image from 8 bit to 32 bit using “Image/Type/32 bit”.

5. Perform “Image/Adjust/Threshold” again and push “Apply” button again. Software will ask “Convert to 8-bit mask or set background pixels to NaN?” We want an image with NAN background pixels, so push “Set to NaN”. Apply for all images.

6. Now, we have created an image with 1 for the ciliary regions, and “NA” for the background. Name this image as “Mask”. Using “Image Calculator” tool, multiply GCaMP6 images with “Mask”. Then, we can get an image with only ciliary regions are extracted.

7. Using the “Polygon selection” tool, register each cilium as ROI. Utilization of ROI manager can be very useful.

8. Using “Image/Stack/Measure stack”, calculate the time course of values. Don’t forget to set values via “Analyze/Set measurements”. For getting mean values, check “mean gray value”.

9. After getting the time-course measurements of values, analyze the data. Generally, we use “R” or “Microsoft Excel” for analysis.

10. For ratiometric analysis, the GCaMP6/mCherry ratio was calculated for each time point. F₀ was measured in the resting state. Then calculate F/F₀ value. An F/F₀ ratiometric change of >2.0 was defined as a peak for our analysis.  

11. The mean frequency denotes the number of spikes per minute, and the peak intensity is the maximum F/F₀ value of each peak. The percentage of cilia showing Ca²⁺ spikes was calculated from the number of ROIs showing peaks and the total number of ROIs examined.

12. Peak duration was estimated with the use of Igor Pro7 (WaveMetrics). Each peak was fitted to an exponentially modified gaussian distribution with the multipeak fitting package, and the peak duration was defined as the width (time) at the position corresponding to 25% of the maximal peak height.

13. Cytoplasmic Ca²⁺ was analyzed similarly. The F₀ value was measured in the resting state, and F/F₀ values were determined from RGECO1 intensity changes calculated from the mean value of all pixels in the ROI.