Some simple macros for Fiji/ImageJ that we are using in the lab.
Most of these tools are available via the quantixed ImageJ update site. Instructions for how to follow a 3rd party update site are here. This is the best way to install these macros and maintain the latest versions.
If you want to install manually, add the contents of macros and scripts to the corresponding directories in your Fiji/ImageJ installation.
After installation, all macros can be found under the menu item called LabCode.
There are macros to help you to make figures with montages - made the way we like them! There are other plugins for making figures in ImageJ but none did what we wanted, so we made our own.
There is a video explaining how to use the Figure Maker here.
Load in your multichannel image, adjust how you want and then crop to a square. To do this select Click Square ROI from the >> on the toolbar.
This tool gives 400 x 400 pixel square ROI, right click to get a different size. Select the area you want.
Crop your images and save them as TIFFs.
To make a nicely spaced montage (row of images). Select LabCode > Figure Maker > Make Montage
You are asked to pick your cropped TIFF.
You can specify the number of grayscale (channel) panels and choose the number of merges.
In the next dialog you can pick which panels go where in your montage and how to merge channels using which colours.
You can also specify a vertical (column) montage rather than horizontal (row).
Grout and scale bars can be added flexibly, no border is added. This is different to ImageJ's Make Montage and makes figure rows the way we like them! Note that if you are going to compile montages, it's best to add a scale bar at this stage to just one of the images.
Your montage is saved in the same directory as the original image. The macro leaves it displayed, so that you can admire your awesome data!
If you want to make many montages, then select LabCode > Figure Maker > Make Montage Directory. This macro allows the user to make montages from all TIFFs in a directory. A dialog is shown at the start that allows the user to select which channels go where and then the montages are made in batch mode.
It is now possible to make a montage of movies or stacks. Select LabCode > Figure Maker > Make Montage and point the dialog at the file you'd like to montage.
If the image is a movie, or a z-stack (but not both) comprising more than one channel, then you will be able to generate a movie/stack montage.
Sometimes, we like to add a ROI and a zoomed version of this ROI to various panels in the montage. To do this open your montage and select LabCode > Figure Maker > ROI Zoom
You can pick which corner you want the zoom and which panels you'd like to add an ROI and zoom.
After clicking OK, you are asked to select the centre of the ROI.
The resulting image is saved in the same directory as the montage.
Note that this code will run on stacks as well so that you can add ROIs and zooms to movies!
It only works on square images and montages made from squares. If your zooms are in the wrong place, check the size(s) of your input images.
When the ROI-Zoom version is made, a text file is saved in the same directory as the resulting image. It contains useful information that you can use to recreate the image if you need to.
Alternatively, you might prefer to highlight the ROI but not insert the zoomed ROI. In this case, Make ROI Zoom External will allow you to save the zoomed ROI as a separate panel. If you'd like to place the zoomed ROI as a further panel, select 0 to zoom the ROI so that it is the same size as a regular panel; otherwise, pick the magnification you'd prefer.
The grayscale panels might look better inverted so that they are black-on-white, rather than white-on black. This can be helpful for dim or small features. To do this, open the montage that you would like to invert and select Invert Montage. This will launch a dialog to help you to choose which panels you'd like to invert and whether you would like to outline the panels with a black border. It's best to do this after you have made the ROI zooms if you want to. There's also an "auto" mode, that will invert all panels except the right/bottom panel (contains the merge) to speed things up.
If you'd like to switch an RGB merge panel for the a different colour scheme, open the montages you'd like to change and select Change Montage Merge. There are some options to switch RGB to OFP (Orange-Fresh-Purple) or to MGB (Magenta-Green-Blue). It is possible to specify any combination of RGB, CMY, W(hite) or OFP. Just select the "Whatever" option and type in your desired combination to map RGB to. This will work on all open images so it is very simple to transform a whole bunch of montages in the same way. It does not work on compiled montages. Note: to run this, you must have NeuroCytoLUTs Update Site installed.
Finally, if we have more than one montage, we need to compile them together. Load in all the montages you'd like to compile. Now select LabCode > Figure Maker > Montage Compiler
The dialog asks you to select which montage you'd like where.
The routine is intelligent enough to recognise vertical and horizontal montages and to compile them left-to-right or top-to-bottom as appropriate.
Again, a text file is saved with the resulting compilation image so that you know which images went where.
Your compilation will save back in the same directory as the montages. Note that you can use this macro to make one compilation and then use it again to add more montages (extra rows) or other compilations. Very large compilation can be built this way.
Note that results from Figure Maker are saved as a flat 300 ppi RGB TIFF ready for use in Illustrator or equivalent software.
Important note about scale bars. The code suggests a pixel size for use in the scale bar. If the scaling of your images is incorrect, then the scale bar will be the wrong size. If the dialog box says 0.069, double-check your original image for scaling information, 0.069 is the "pixel size" for a 300 ppi image, i.e. the scaling no longer relates to the original image. Bottom line is: make sure you know the pixel size of your images before adding any scale bars.
For multichannel microscopy images, e.g. from an immunofluorescence experiment, quantixed follows these rules for best practice.
- Individual channels as grayscale - reason: the eye does not detect black-to-red in the same way as black-to-green or black-to-blue
- In a row montage the merge is on the right. In a column montage it is at the bottom
- Square images, square ROIs and square zooms
- No border
- Scale bar in the bottom right corner (added to the compiled montage)
- Fixed grout of 8 pixels (suggested)
- Scale bar of 10 µm, height of 2 x grout (suggested)
- Grouting between conditions is 2 x grout between channels (suggested)
- Labelling is done in Illustrator or some other software to assemble the final figure, not in ImageJ
The basic workflow is to go:
IMAGE(S)
|
| (Montage Maker or Montage Maker Directory)
v
MONTAGE(S)
|
| (Montage Compiler)
v
COMPILED MONTAGE
Images can be XYC, XYCT or XYCZ. The Montage is save as RGB and will be XY, XYT or XYZ, respectively.
Montages can be modified in the following ways:
- ROI zoom or ROI zoom external - can be 1 or more times
- Inverted
- Change colors
These steps can be run iteratively, e.g. ROI zooms twice, then invert, then change colors. Montages and modified montages can be compiled together as long as the dimensions are compatible.
ROI Zoom macros and Compile Montage require images open to work with, the other macros typically do not allow open images, and you need to find the source image upon running.
Check whether your figure or image is colour blind safe using this simple utility Check_Colour_blindess.ijm.
BlindAnalysis.ijm Takes a directory of TIFFs, strips the label from them and saves them with a blinded name. A tsv called log.txt is created to log the association between the original file and the blinded copy. Works on TIFF only.
This is a simple utility to save a version of the file you are viewing in Fiji/ImageJ to put in your electronic lab notebook. A png version or low-res movie of the file you are looking at is saved to the Desktop with a unique name. A text file is saved to explain the filenames.
Two macros that will:
- Add arrows to movies to highlight and track an object of interest. Unlike other scripts out there, this one places an arrowhead in a fixed orientation/distance from the object and allows annotations in any frame (not all frames).
- Irregular Time Stamps - adds timestamps to movies, formatting them in hh:mm or mm:ss. Unlike other versions, negative times are allowed and the time intervals do not have to be regular. Requires a new line separated text file of times.
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In qUtils there are some other utility macros that we use in the lab.
We've had problems with nd2 files being saved with a LUT that is not pure red, pure green or pure blue. This is because it uses a LUT that is based on absolute wavelengths of the fluorophore. You can use Make_NIS_Pure_RGB.ijm to sort out the colours.
Open all the nd2 files in a directory and save them as TIFF to another directory with nd2SaveAsTiff.ijm.
Maybe you like to open a whole directory of images, look through them, closing the bad ones and leaving the good ones open. Perhaps you want to grab the list of good images so that you can come back to it later? Well, PrintTitlesOfNiceImages.ijm does this for you.
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Most problems are solved by allowing Fiji's updater to install the latest code from the quantixed ImageJ Update Site.
Still not working? All routines use a number of custom functions which get loaded when Fiji starts up. Errors in running these routines usually traceback to third party code that modifies StartupMacros.fiji.ijm. Which can be found in the macros directory. Specifically, if the third party has deleted this code block, then none of the autorun functions happen at startup.
macro "AutoRun" {
// run all the .ijm scripts provided in macros/AutoRun/
autoRunDirectory = getDirectory("imagej") + "/macros/AutoRun/";
if (File.isDirectory(autoRunDirectory)) {
list = getFileList(autoRunDirectory);
// make sure startup order is consistent
Array.sort(list);
for (i = 0; i < list.length; i++) {
if (endsWith(list[i], ".ijm")) {
runMacro(autoRunDirectory + list[i]);
}
}
}
}
If you can't bear to uninstall the third-party code (or don't know which update site causes the problem), just paste that code block into StatupMacros.fiji.ijm and things will start to work again.
Other notes Note that usage of RGB images has a limitation of a filename length of 59 characters.
The grout looks really tiny and the scale bar is very thin The suggested grout sizes work well for 400x400 images. If you have 1000x1000 images the suggested grouts will look thin when imported into a figure, adjust accordingly. As a guide, a 600x600 image that has two panels and one merge with a grout of 8 pixels will be 1816 pixels wide. At 300 dpi this is a final size of 155 mm. A full size figure is usually 170 mm, so the figure will be ~91% of the full width of the figure. If you shrink it to half size, now the grout will be 4 pixels or 0.34 mm which will look quite thin. A rule of thumb is grout should be 1/50th of the image width, but ultimately, test it out!