Fluorescence Analysis with ImageJ and Excel
How to calculate ΔF/F₀ from a series of fluorescence images

This page describes how to analyze fluorescence intensity (ΔF/F₀) from a series of images using ImageJ and Excel. While semi-automated custom tools are often used to facilitate rapid analysis of fluorescence data, there is great value in understanding how to realize these analyses using standard and commonly-available software.

Measure ROI Fluorescence

  • Install ImageJ (Fiji)
  • Drag/drop a folder of images onto ImageJ to open all images as a stack
  • Create a maximum projection (of either channel) to make drawing of ROIs easy. Image > Stack > Z project and select Average (not maximum projection!)
    • do all ROI creation on the projection. You can later use these same ROIs on the original data.
  • Click analyze > tools > ROI manager (ROI means “region of interest”)
  • Use the “freehand sections” tool (as opposed to the square or circle) to outline some cells.
    • Every time you outline a cell, click the add button on the ROI manager.
    • I recommend naming cells as you add them (click the rename button).
    • Be sure to indicate whether the thing is a neuron or astrocyte.
  • When all cells have been outlined, highlight them all (with shift-click or control-click) and save the ROIs by clicking more > save.
    • once ROIs have been saved, you can load the same ROIs on the red stack, green stack, or a projection image
  • Deterime what data to analze by clicking analyze > set measurements
    • I recommend selecting ONLY “Mean gray value”
  • Analyze the data in the ROI manager by clicking more > multi measure
  • In the results window, click file > save as and call it something
    • Excel 2016 may not be able to open the .xls file. You may have to rename it to .tsv (short for tab-separated values) and drag/drop it into a blank excel worksheet.
  • Once the data is in excel, add a “time” column and populate its values appropriately

Calculate Single-Channel ΔF/F₀

Single-channel fluorescence experiments report the change in fluorescence (ΔF) normalized to its baseline level (F₀). In this example F is measured for each ROI in every frame. The following steps are performed for each ROI.

  • Calculate F₀ as the mean fluorescence intensity during the baseline region

  • Calculate ΔF by subtracting F₀ from every F value

  • Calculate ΔF/F₀ by dividing ΔF values by F₀

Calculate Ratiometric ΔF/F

The following describes how to measure ratiometric fluorescence from a series of 2D multi-channel images. See Ratiometric Linescan Analysis with ImageJ and Excel for information specific to analyzing ratiometric linescan images.

Two-channel fluorescence experiments report ΔF/F as the change in one fluorophore relative to another. In this example we will use G to represent a calcium-sensitive fluorophore and report the change in its fluorescence relative to a calcium-insensitive fluorophore R. In this example G/R is measured for each ROI in every time point. The following steps are performed for each ROI.

In this case F represents G/R so ΔF/F is: [Δ(G/R)]/(G/R)₀

  • Calculate (G/R)₀ as the mean ratio of fluorescence intensity during the baseline region

  • Calculate Δ(G/R) by subtracting (G/R)₀ from every (G/R) value

  • Calculate ΔF/F by dividing Δ(G/R) values by (G/R)₀