ImageJ macro for analyzing fluorescence bleach curves obtained from 96 wells plates
Runs on 2019 FIJI and ImageJ The macros were tested on MacOS Mojave (10.14.5) running: ImageJ v1.52p with Java 1.8.0_101 (64-bit) and on Fiji Version 2.0.0-rc-69/1.52p.
The macros were tested also on Windows 10 (version 1803 for x64-based systems) running: ImageJ v1.52p with Java 1.8.0_112 (64-bit) and on Fiji version 1.52p running with Java 1.8.0 172 (64-bit)
BioFormats v.6.1.1 or v 6.1.0 (https://www.openmicroscopy.org/bio-formats/) must be installed in ImageJ. In Fiji this is installed automatically.
For usage see main manuscript Secondary screen - Photostability in mammalian cells.
ImageJ & FIJI macro's can be dragged and dropped on the toolbar, which opens the editor from which the macros can be started. Macros can also be loaded via Plugins->Macros menu, either use Edit or Run.
Test can be downloaded from following zenodo repository :
- Work on current image or load from directory: Here you can choose to either use the current (e.g. a hyperstack with 96 positions and n time steps) image already displayed in ImageJ, or you load (a) file(s) from a directory. The latter can be a series of timelapse stacks in one directory or a hyperstack image with for instance 96 or 384 positions and n time steps.
- 96 wells or 384 wells: here the well plate format can be selected.
- Fixed threshold value or modal value threshold: Here you can choose how cells are recognized in the first time point image of the bleach series, either by selecting a fixed threshold intensity above which you assume there are cells, or a modal value determination that determines the modal (background) grey value and uses a statistical evaluation of pixels above this background.
- In case of fixed threshold, what intensity over the background: in case the previous choice was fixed, this is the lower intensity threshold for selecting cells in the analysis, otherwise this is a dummy input.
- Lower Threshold=number x Stdev + modal: In case a modal threshold was chosen for analysis, this value sets the lower intensity threshold for analysis based on the modal value + this input times the standard deviation found in the image. In case a fixed intensity threshold is chosen this is a dummy input.
- Keep cell ROIs: if selected an output image stack is generated with all analyzed cell ROIs per well.
- Create output 96/384 well tau image: if selected a colored bleach time to 50% of initial intensity image is generated.
- Low/high threshold for 50% time (2x): sets the minimal/maximal 50% bleach time for display in the colored bleach time multiwell image.
- Automatic determination of thresholds: If selected, the previous 2 inputs will be overruled and the macro will scale the tau multiwell output image according to the minimal and maximal measured 50% bleach times.
- Create output 96/384 well non-bleached image: If selected, a multiwell image is added of the percent of initial intensity remaining in the last point of the acquired bleach time series.
- Create output 96/384 well initial intensity image: If selected, a multiwell image is added of the detected average initial intensity. This is useful for inspecting wells with very bright or dim cells.
- Start row/Column: In case not an entire 96 well or 384 well is screened but a subsection of the plate, the first well (row, column) can be chosen. In case a 24 well plate is used, a 24 well plate output can be made by selecting E7 as first well.
- Acquisition in meandering mode: If selected the sequence of bleach images is assumed to be in the order A1-A12, B12-B1, C1-C12, D12-D1, E1-E12, F12-F1, G1-G12, H12-H1 for a 96 well plate. If not selected it assumes an order A1-A12, B1-B12, C1-C12, D1-D12, E1-E12, F1-F12, G1-G12, H1-H12.