NoiSee - An easy-to-use ImageJ macro for measuring SNR (signal-to-noise ratio) of light microscopes.
Enable the NoiSee update site and restart Fiji.
NoiSee consists of two independent macros, one for each type of analysis:
- The Beads Method for a time-lapse acquisition of fluorescent beads. NOTE: this method will NOT work for sub-resolution beads! The beads should have a diameter of at least ten pixels in the acquired image.
- The Fluorescein Method for an acquisition of a fluorescent solution and a dark field (i.e. "laser off") image, both single frame (no Z-stack, no time-lapse).
Type "NoiSee" in the quick search bar, select "Bead Analysis" and click "Run". Otherwise, you can also locate the NoiSee macros in the Plugins > NoiSee menu.
First, the parameters dialog will appear with the following input fields / options:
- In the image field specify the path to a time lapse beads image stored in a format readable by Bio-Formats.
- All result files generated by NoiSee will be placed in a new sub-folder located in the same directory as the input image.
- The approximate beads diameter has to be specified in pixels. NoiSee uses the "Find Maxima" function to automatically detect beads above a certain intensity threshold that needs to be roughly estimated. Starting values for this estimate depend on the detector type used, e.g. 50 for PMTs, 10 for Hybrid detectors in photon counting mode or 500 for camera based systems.
- If not all beads are identified the estimate should be lower.
- If peaks other than beads are identified the estimate should be higher.
- Note: While NoiSee tries to exclude hot pixels and overexposed beads, these would ideally be already avoided during imaging.
- Choose whether you wish to create kymographs. We recommend to do so as they are useful to inspect sample drift and bleaching.
- NoiSee can further save additional data (individual measurements per bead) as txt-files.
- All images and plots created by NoiSee can optionally be saved in a PDF report.
- ROIs used for measurements are available from the ROI manger and can additionally be visualized as extra images.
- Press "OK" and wait for NoiSee to finish its calculations.
- A summary of all NoiSee results is automatically saved as a txt-file.
Please note that NoiSee is based on a publication. If you use it for related research please be so kind to cite our work:
Alexia Ferrand, Kai D Schleicher, Nikolaus Ehrenfeuchter, Wolf Heusermann, Oliver Biehlmaier: Signal-to-Noise ratio made easy: A tool to assess your confocal performance bioRxiv 291500.