Image stack axis calibration

Image stack axis calibration assigns physical coordinates to the indices of an image stack. It states what each axis means, its spacing, origin and orientation. Pixel indices, section indices and frame indices are distinct from positions and times. In ImageJ, image properties include pixel width, pixel height, voxel depth and frame interval; its Origin fields are expressed in pixels.^[ref]

For an original general spatial model, let the index vector be $\mathbf{n}=(i,j,k{)}^T$ and definewhere ${\mathbf{r}}_0$ is a physical origin and $R$ is a rotation matrix. This is an affine map; the rotation represents a chosen acquisition-to-world orientation rather than a claim that ImageJ's Properties dialog implements an arbitrary rotation. Convert any pixel-unit origin into the chosen physical convention before using this equation.

Take spacings $(s_x,s_y,s_z)=(0.2,0.3,1.0)\mu \mathrm{m}$, origin ${\mathbf{r}}_0=(10,-4,2)\mu \mathrm{m}$ and a 90° rotation about the positive third axis:Index $(3,2,1)$ first maps to local displacement $(0.6,0.6,1.0)\mu \mathrm{m}$ and then to world position $(9.4,-3.4,3.0)\mu \mathrm{m}$. Its distance from the physical origin is $\sqrt{0.6^2+0.6^2+1^2}\simeq 1.31\mu \mathrm{m}$, unchanged by the rotation.

Cropping and axis permutation require corresponding metadata changes. If a crop begins at old index $\mathbf{c}$, its new physical origin is ${\mathbf{r}}_0^{\prime }={\mathbf{r}}_0+R\mathrm{diag}(s_x,s_y,s_z)\mathbf{c}$. For irregular section positions, store those positions explicitly instead of inventing one uniform depth increment. Image resampling may alter the grid, but it does not create independently acquired depth information.