With a stack or hyperstack, only a single 2D slice is 'active' at any one time. Extra sliders at the bottom of the image window are used to change which slice this is (Figure 2). In the case of multichannel images, any changes to lookup tables are only made to slices of the currently-active channel.

The dimensions of an image can be seen in the top entries of Image ▸ Properties…​. Occasionally these can be incorrect: perhaps different z-slices were wrongly interpreted as time points when a file was opened, or the presence of multiple channels was not spotted. This can affect not only the display, but also some processing or measurements. Fortunately, dimensions can be corrected manually using the command Image ▸ Hyperstack ▸ Stack to Hyperstack…​ – provided you know, or can work out, the right values.

It is helpful to consider the pixels of 3D data as being densely packed into a transparent box that could be viewed from different angles (Figure 3A). Visualizations like this can be made with Plugins ▸ 3D Viewer. They are particularly good for generating attractive figures or impressive movies, but details can be hard to interpret because they are influenced by perspective and which pixels overlap from our current viewing angle.

To systematically explore data, therefore, it is usually preferable to look inside the box by generating 2D images from only 3 angles: from above (xy) and from two remaining sides (xz and yz). These 3 viewpoints are orthogonal (i.e. they are oriented at 90 to one another), and the command Image ▸ Stacks ▸ Orthogonal Views makes this easy. It opens up 2 extra windows, so that when you click at any point on the original xy view, you are shown cross-sections through that point from each direction.

Another extremely useful way to collapse the data from 3 dimensions into 2 is to use a z-projection. The basic idea is of taking all the pixels in a stack that have the same x and y coordinate, applying some operation to them, and putting the result of that operation into a new 2D image at the same x and y position.

Two important operations in fluorescence imaging are to add all the pixels with the same xy coordinates (a sum projection), or to compare the pixels and select only the largest values (a maximum projection), both implemented under Image ▸ Stacks ▸ Z Project…​. The advantage of the first is that every pixel value has an influence on the result: which is good if you plan to measure intensities in the projection image (although quantitative analysis of projections can be somewhat dangerous, e.g. if intensity measurements are compared between projections made from stacks with different numbers of slices). The advantage of the second is that it tends to give a nice and sharp looking image, since structures are at their brightest in the planes where they are in focus (Figure 4B). Naturally, you could make a minimum intensity projection if you liked, but a very out-of-focus-looking image is generally less desirable (Figure 4C).