joachimgoedhart/quantify-intensity-from-timelapse

Step-by-step instruction to quantify fluorescence intensities from timelapse imaging experiments

Quantify Intensity from Timelapse

Step-by-step instruction to quantify fluorescence intensities from timelapse imaging experiments using open source software (ImageJ and PlotTwist)

Preparations

Install the ImageJ macro file "Subtract_Measured_Background.ijm"

• Download the ImageJ macro file "Subtract_Measured_Background.ijm" (available in this repository)
• Install it as a plugin: Plugins > Install Plugin...
• The Plugin is installed in the drop-down menu Plugins as Subtract Measured Background.

Set Measurements

• Set the measurements to only measure mean gray values: Analyze > Set Measurements... select Mean gray value

Set the correct options for export

• Set the options for input/ouput: Edit > Options > Input/Output...
• Set the "File extension for tables" to .csv
• In the "Results Table Options" make sure that "Save column headers" and "Save row numbers" are selected

Analysis

Open the image sequence

• If the images are individually saved (as in the example data) use: File > Import > Image Sequence...
• If the images are saved as a single file, use the appropriate file import option

Define and subtract background

• Use an ROI to define an area with background fluorescence in the image
• Use Subtract Measured Background to remove the average value of the ROI from the image (this action will be performed on each image in the stack)

Select and analyze cells

• Set the measurements to only measure mean gray values: Analyze > Set Measurements... select Mean gray value
• Activate the ROI manager: Analyze > Tools > ROI Manager...
• Draw an ROI in the image sequence to select a cell (try to exclude background), add to ROI manager ("Add [t]")
• Repeat the selection of cells with ROIs until all cells are marked
• Save the set of ROIs (More... > Save...)
• Select all ROIs in the ROI manager
• Measure in all ROIs the mean gray value: More > Multi Measure (select "Measure all slices"" and "One row per slice")
• Save the table with results (csv format).

Plot the result

• Open the csv file with results from the quantification in PlotTwist by using the "Upload" option
• PlotTwist has the option to apply normalization of the data
• PlotTwist has the option to exclude data from user selected cells

Sample data and expected results

• Raw sample data is available in this folder
• Background corrected data together with the ROIs and the results of the measurement is available in this folder
• The graph of the example data generated with PlotTwist looks like this:

(FRET) ratio analysis

In case of emission ratio imaging, two intensity traces are acquired. The traces are acquired at different emission wavelengths (In case of FRET ratio imaging, one is acquired at the emission wavelength of the donor and another acquired at the acceptor wavelength). Both intensity traces need to be background corrected (according to the procedure describe above under Define and subtract background). Next, a new stack with the ratio data can be obtained in ImageJ:

• When both background corrected image stacks are open, choose Process > Image Calculator...
• Select the stacks with emission data, for instance "Image1:" stack acquired at the first wavelength, "Image2:" stack acquired at the second wavelength
• Choose the operation "Divide"
• Make sure that the checkboxes "Create new window" and "32-bit (float) result" are activated.
• The result of the operation will be a new stack with emission ratio data.
• The ratio data in this stack can be quantified for each cell as described above under Select and analyze cells