Ratiometric Linescan Analysis with ImageJ and Excel
How to calculate ΔF/F from ratiometric linescans using ImageJ and Excel

This page describes how to analyze fluorescence intensity (ΔF/F₀) from a ratiometric linescan using ImageJ (Fiji) 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 Fluorescence with ImageJ

  • Rotate images so time proceeds horizontally from left to right.
    • If original data is vertical (time proceeds top to bottom), Image > Transform > Rotate 90 Degrees to Left
  • Merge G and R into a composite image
    • Click Image > Color > Merge Channels
    • I recommend making the red channel magenta rather than red
  • Draw a ROI around the structure of interest in the composite image
  • Click Analyze and select Plot Profile
  • Click Live and notice the curve change as you change channels using the scrollbar in the composite image
  • For each channel, click Data, select Copy All Data, and paste the result in Excel
  • The first column (pixel number) should be replaced by times

💡 The amount of black space captured by the ROI only slightly affects the resulting ΔF/F because fluorescence intensity in each channel is summed before taking the ratio. Note that mean ROI intensity is just the sum divided by a fixed area. Summing fluorescence from extra blank space does not affect the result beyond contributions from noise and basal fluorescence.

Calculate Ratiometric ΔF/F with Excel

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

Sample Data

This excel file: linescan.xlsx

Images analyzed:

Green Red


  • ROI Analysis Pipeline (GitHub) is a collection of software tools for analyzing fluorescence images

  • ScanAGator (GitHub) is a tool that facilitates analysis of ΔF/F from ratiometric linescan images