"ImageJ is a public domain Java image processing and analysis program inspired by NIH Image for the Macintosh. It runs, either as an online applet or as a downloadable application, on any computer with a Java 1.5 or later virtual machine. Downloadable distributions are available for Windows, Mac OS X and Linux. It can display, edit, analyze, process, save and print 8-bit, 16-bit and 32-bit images. It can read many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and ‘raw’. It supports ‘stacks’ (and hyperstacks), a series of images that hare a single window. It is multithreaded, so time-consuming operations such as image file reading can be performed in parallel with other operations ."
(read all about it: here)
Download the free Fiji software from here picking the release that is appropriate to your computer system.
You can open your data with a range of methods.
The most common are File > Import > Raw for volume (voxel) data and File > Import > Image Sequence for image stacks. For the former, you will need to know the dimensions of your data (X, Y, Z and bit-depth). Image sequences already 'know' their x and y dimensions, and bit depth, and the number of images in the sequence defines the z size. If you have a .vgi or .vgl file, opening this in a text editor such as Notepad or TextEdit will let you see what these dimensions are. For more details read below:
To import a .raw file navigate to File menu and pick Import > Raw. You will then be prompted to enter information about the volume, which (in general) can be found in the filename. The generic XRH filename format is:
<DATE>_<MACHINE>_<JobID>_<Operator or Researcher Initials>_<XRHSampleID>_<comments>_<X-dimension>x<Y-dimension>x<Z-dimension>x<bit-depth>.raw
for example for a volume of 2000x2000x200, 16-bit voxels, the filename might look something like
20200501_XRH_1234_ABC_SXAA12345-FFPE_Sample3_2000x2000x200x16bit.raw
N.B. If your workstation does not have enough RAM to load the whole volume then the Use virtual stack option should be used. This allows opening data sets larger than the available RAM, with some performance penalty.
When using more than 8-bits, if the image appears incorrect, please try checking (or unchecking, if checked) the Little-endian byte order option.
In most of cases .vol files will be accompanied by a .vgi file, which contains all the information you need to import the .vol. The .vgi can be opened with any text editor (e.g. right click > open with > TextEdit).
Fields of interest here are: size, bitsperelement and resolution. For example if:
.vgi fields are:
size = 1500 1400 1800
bitsperelement = 32
resolution = 0.00945568
Fiji/ImageJ Import fields should be:
Width=1500, Height=1400, Number of images=1800
Image type 32-bit Real
That's the pixel/voxel size in mm. You can use this to calibrate your images; see set Scale
You can import some video files using the File > Import > Movie (ffmpeg) option. For this you will need to make sure that the FFMPEG repository is installed (see "Plugins and repositories" > Activate FFMPEG repository below).
Using File > Save As > Raw Data or File > Save As > Image Sequence are the most common ways to save your data. Take care not to overwrite any original data, as it may be difficult (or impossible) to recover this.
When saving volume as .raw, make sure you take note of the volume size and bit-depth (see: Importing .raw volume files above). We highly recommend to include this information in the filename following the generic µ-VIS naming convention (see: Importing .raw volume files)
Any conversion of this nature (e.g. 32-bit to 8-bit, 16-bit to 8-bit) can be performed in Fiii/ImageJ.
If you have a volume file, open it with File > Import > Raw. If it is an image stack, use File > Import > Image Sequence.
After your data has loaded, use the inbuilt brightness and contrast processing tools (Image > Adjust > Brightness/Contrast) to ensure that you have adjusted the contrast around the levels of interest. Typically, selecting Auto will be good enough. You can scroll through your dataset in ImageJ to verify this. Note that each slice will have a slightly different behaviour when pushing Auto.
Once you are happy with the results, you can then go to Image > Type > 8-bit, and the conversion will begin. You now have an 8-bit dataset.
If you wish to save this dataset so that you can work with it in a differnet software (e.g. Dragonfly), then use
File > Save As > Raw Data... or
File > Save As > Image Sequence... or
File > Save As > Tiff...
Go to the Image > Adjust menu and select Threshold. This will bring up a box around a histogram representing the lower and upper thresholds. Change the colour from Red to B&W.
You can adjust the two sliders just below the histogram to control the lower and upper bounds of the thresholding. With the B&W setting, anything within the bounds of the threshold will appear black, and anything outside will appear white. To invert this, select Dark background.
When you are happy with the results, click Apply and close the thresholding window.
There are two straightforward methods for this:
Method 1 : Go to Edit > Invert, and confirm the warning dialogue that comes up to process all images.
Method 2: Use the LUT inversion tool at Image > Lookup tables > Invert LUT to reverse the colour schemes lookup table. In the case of a greyscale image with a sequential lookup table, this will replace whites with blacks, dark greys with light greys and vice versa.
Plugins in ImageJ often come from third parties, and the precise implementation of any interface may vary from developer to developer. Fiji is a version of ImageJ that has a large set of pre-installed plugins. Plugins can be installed manually by copying the relevant files into the plugins folder and restarting Fiji/ImageJ or by activating the relevant repository (when applicable).
Repositories can be installed by activating the respective update site. To do this navigate to Help > Update... (N.B. not Update ImageJ...). From there, click on Manage update sites and select the repository you want to install.
To install the FFMPEG plugin which allows import and export of movie files activate the FFMPEG repository.