Comparative analysis of 2D and 3D distance measurements to study spatial genome organization

- 2D and 3D measurements yield similar results in flat cells and at long distances.
- 2D measurements provide more precise data.
- 3D measurements provide more accurate data, but are noisier.
- To gain precision in 3D distances, it is crucial to sample at 1/3 the depth of field.

The spatial organization of genomes is non-random, cell-type specific, and has been linked to cellular function. The investigation of spatial organization has traditionally relied extensively on fluorescence microscopy. The validity of the imaging methods used to probe spatial genome organization often depends on the accuracy and precision of distance measurements. Imaging-based measurements may either use 2 dimensional datasets or 3D datasets which include the z-axis information in image stacks. Here we compare the suitability of 2D vs 3D distance measurements in the analysis of various features of spatial genome organization. We find in general good agreement between 2D and 3D analysis with higher convergence of measurements as the interrogated distance increases, especially in flat cells. Overall, 3D distance measurements are more accurate than 2D distances, but are also more susceptible to noise. In particular, z-stacks are prone to error due to imaging properties such as limited resolution along the z-axis and optical aberrations, and we also find significant deviations from unimodal distance distributions caused by low sampling frequency in z. These deviations are ameliorated by significantly higher sampling frequency in the z-direction. We conclude that 2D distances are preferred for comparative analyses between cells, but 3D distances are preferred when comparing to theoretical models in large samples of cells. In general and for practical purposes, 2D distance measurements are preferable for many applications of analysis of spatial genome organization.