Documentation

Regarding bugs and errors

We regularly release new versions to fix known bugs.

If you are experiencing errors, make sure to update to the latest version by downloading and installing Fluidity Calculator again.

If this doesn't solve your problem, please reach out! We will be happy to help.

Fluidity Calculator

Fluidity Calculator is an ImageJ macro tool that works in Fiji. It accompanies this protocol paper where we describe how to quantify membrane fluidity using fluorescence microscopy and membrane probes.

Fluidity Calculator calculates the GP parameter of each pixel based on the following equation:

$$\text{GP} = \frac{\text{Ch1} - \text{Ch2}}{\text{Ch1} + \text{Ch2}}$$

Here is an explanation of what each parameter in the GUI does:

• Channel 1: This is the label (wavelength) of the spectral channel that will be assigned to Ch1, the ordered (blue-shifted, lower wavelength) channel. If 'Channel Window Range' is larger than 1, this wavelength will be in the middle of the range.

• Channel 2: This is the label (wavelength) of the spectral channel that will be assigned to Ch2, disordered (red-shifted, higher wavelength). If 'Channel Window Range' is larger than 1, this wavelength will be in the middle of the range.

Advanced Options:

• Channel Window Range: This is the number of channels that will be combined to generate Ch1 and Ch2. When we obtain spectral images, each channel corresponds to a narrow emission window. By combining those narrow channels we can increase our signal at the expense of GP resolution.

  • For example: Let's have a look at the 'NR12A – POPC.lsm' file in the 'sample images' folder. This is a spectral image consisting of 8.9 nm channels and starting at 503 nm (503, 512, 521, 530...). If we choose:
    • Channel 1: 592 nm
    • Channel 2: 646 nm
    • Channel Window Range: 3
  • Then the tool is going to combine the following signals:
    • Channel 1: 583, 592 and 601 nm
    • Channel 2: 637, 646 and 655 nm

• Mean Filter: This is a smoothing filter meant to make thresholding more consistent. It smooths the image by replacing each pixels with the mean of those in around it (more info: https://imagej.net/ij/docs/menus/process.html#filters).

  • It is important to note that the filter is only applied to the reference threshold image, but never to the raw data. The GP will be calculated based on the raw date.

• Mean Filter Radius: This parameter influences the strength of the mean filter. The higher the value the smoother the image will look at the expense of losing resolution.

• Thresholding Channel: Thresholding can be performed in either of the channels defined in the previous steps or in the sum of both. We recommend using Channel 1+2 to increase the signal-to-noise ratio. ‘Channel 1’ and ‘Channel 2’ can be used in specific situations, for example to threshold out internalised signal. However, it must be used with caution, as it can greatly biased the obtained GP values.

• Saturated Pixel Warning %: If the image contains more than the set value of saturated pixels after thresholding, the tool will display a warning message. It will also allow the user to see which pixels are saturated. Note that saturated pixels must not be used for GP calculation and are always removed from the analysis.

• Calculate Emission Spectrum: It will show the emission spectrum of the thresholded pixels. In the case of time lapse or z stack images, it only supports single frame/slice. The user will be asked to select the frame/slice. However, if the results are saved, the spectrum of all slices and channels will be included in the CSV file.

Save Options:

• Save Results: When active, the following will be saved:
  • GP Image in tiff format. Open with ImageJ, most programs do not display this properly.
  • GP_Results table. It includes the median, mean, standard deviation and average signal of each channel (before threshold).
  • Histogram image: The GP histogram in tiff format. It will only display a single frame/slice in the case of time lapse and z stacks.
  • Histogram table: The GP histogram data in csv format. It includes the data for all frames/slices.
  • Parameters table: The parameters set by the user that allow to replicate the analysis.
  • Spectrum table: It is only saved if ‘Calculate Emission Spectrum’ is active. The intensity on each spectral channel in csv format. It includes the data for all frames/slices. The analysis might take a bit longer, especially in the case of large images.
• Save Analysis Mask: Only applies if ‘Save Results’ is checked. To save the image showing which pixels have been considered for GP analysis. It can be useful for downstream analysis, for example, to correlate the fluorescent signal of a different fluorophore with the GP data.
• Save to: The directory where the result images/tables will be saved.
• Save as Default Settings: The next time the tool is run, it will set the used settings as default.