Image Analysis in Fiji
Saving Time Series and a Time-lapse Move
1. File>Import>Image Sequence... 2. Import image sequence as a virtual stack. 3. File>Save As...>AVI to save as a movie file that is readable across devices. Here you have lots of options. Consider the frame rate that you would like (this depends on the number of images you have). Typically I compress so that 1 second represents 1 hr of real time. This would be 60fps if you took one image every minute, and 30fps if you took 1 image every 2 min. This may take a while to save. 4. Other options are available if you want a smaller video file, including making sure you are saving 8bit versus 16 or RGB images. You can also create a substack of, for example, every 10th frame and save that as an AVI.
Time Series Image Analysis
1. Import your image sequence into Fiji using a virtual stack. 2. Convert to 8-bit grayscale by using Image>Type>8-bit. This might take a while using a virtual stack. 3. Once all images have been converted to 8-bit, run the Stack Difference command, which will subtract pixels in one frame from the pixels in the previous frame. Analyze>Multi Kymograph>Stack Difference. You can start with a frame difference of 1 and adjust if interested. This should bring up a new window of mostly dark pixels (adjust so that it is visible with the brightness and contrast settings). You can save this as an AVI as well if you choose. 4. Open the Time Series Analyzer plug-in (needs to be downloaded and installed if this is your first analysis) and draw a rectangular ROI around the entire frame or region you are interested in. You can make multiple ROIs as well. Press 't' to add your ROI to the ROI manager, then highlight it and "Get Average" on the Time Series Analyzer window. This should bring up a plot of the average pixel intensity across your ROI over time. Check different areas, compare mutants to WT, etc. Evidence of pulsing tends to look like sharp peaks with a period of ~45-60 min that occur after fruiting bodies have formed.
You may need to experiment with smoothing techniques or the Walking average function to smooth some of the noise first.