Intensity Over Time

Introduction

Live imaging can provide a wealth of information that cannot be achieved form fixed samples alone. In this technical note you will be shown how to measure intensity spikes in a calcium flux experiment and to measure the contractility of some live tissue but the principals demonstrated can be applied to any time series analysis.

Single Point Analysis

1. Open Calcium Flux.tif from the Demo Images\Widefield\Calcium Flux folder. 

If you play through the stack you will notice that the cells are flashing and flashing at different rates. 

2. To measure one point draw a ROI (a circle in this example) on the area you want to measure

3. Before measuring we need to reconfigure the Set Measurements options to only show Mean Grey Level. 

4. No go to Image 🡪 Stacks 🡪 Plot Z Axis Profile. The output will be a trace graph of the intensities and a table of the raw values that can be saved for later. 

5. Move the ROI to a different cell and plot the profile again. Notice that different cells have different responses. 

6. Also try moving the ROI to the background where there are no cells and plot the profile. 

While the scale of the spikes is very low (13 vs. 250 grey levels) they need to be taken into consideration when calculating final values.

Multi Point Analysis – Manual ROI Entry

Analysing single points at a time is ok if there are not too many, but if you need to measure many points there is an alternative.

1. Close all the result and graph windows so you are only left with the original calcium flux image. Go to Analyse 🡪 Tools 🡪 ROI Manager 

2. Now draw your first ROI on the calcium flux image. Once you have it drawn press the Add button in the ROI Manager. The ROI will be added to the list. 

3. If you want to give it a more meaningful name just press the Rename… button and type in what you want. 

4. Add more ROIs by holding down the shift key and then adding (and renaming them) to the ROI Manager. Don’t forget to include one for the background. 

5. Tick the Show All box. Then click on the More>> button and select Multi Measure. 

6. Make sure both boxes are ticked in the next window and press OK. 

7. You won’t get graphs this time, just a results table that can be saved and graphed later in Excel. 

Multi Point Analysis – Automatic ROI Entry

If the image has the right information in it, or there is a separate channel that marks out the cells, the ROIs can be made automatically by segmenting the cells out.

1. Open Calcium Sensing.tif from the Demo Image\Confocal\Calcium Sensing folder.

If you play through the stack you will notice that the cells stack in one place so we don’t need a secondary channel to be able to measure the cells automatically.

2. Create a maximum intensity Z Projection of the stack. 

3. Smooth the projection, threshold it and make a binary image. 

4. Some of the cells have joined together so use the Binary Watershed function to separate them. 

5. Configure the Analyse Particles module to create a mask of the individual cells, remove edge cells and small debris. Also make sure the Add to Manager box is ticked so we can save the ROIs created. 

6. Save the ROIs and close the binary mask image. 

7. Load the saved ROIs onto the original image stack. 

8. Manually draw a ROI on the background and add it to the ROI list. 

9. Use Multi Measure to get the results for all the ROIs. Once again, the results can be saved and graphed in excel later. 

Tissue Contractility

Live imaging of tissues or organoids can provide useful information of that samples response to environmental changes or drug responses. An easy way to measure this is to measure the frequency of contractions in the sample. The sample does not need to be fluorescently stained, as long as there is some intensity fluctuation this type of analysis will work.

1. Open Contractility.tif from Demo Images\Widefield Images\Tissue Contractility

2. Use the Line ROI tool to draw a line across the edge of the tissue where it moves across the background 

3. Go to Image 🡪 Stacks 🡪 Plot Z Axis Profile to generate a graph showing the fluctuations in intensity that can be used to measure the frequency. You can press the Live button on the graph to see it live update if the ROI is moved. 

4. As before you can press the List button to generate a table that can be used to graph and analyse the data in another program.

5. There is the option to do some simple analysis of the graph in Fiji using the BAR tools. Have the graph selected and go to BAR 🡪 Data Analysis 🡪 Find Peaks 

6. The Find Local Maxima/Minima dialog will let you configure what you want to call a peak. A calculated value is automatically put in the Min. peak amplitude box, it is the Standard Deviation of the intensities measured. You can use the other options to filter out peaks you don’t want to include. For example, you may want to set a value for Min. value of maxima to exclude peaks that are close to background. 

7. Press OK and the peaks and troughs of the graph will be detected and counted. Pressing List in the new graph will give a results table that contains the values of the minima (blue) and maxima (red) circles that are drawn on the graph.