Figure 1.

Three models of water movement could explain the decrease of extracellular volume fraction and tissue shrinkage observed in the EM image stack and raw reconstruction. Morphological changes to the extracellular space (ECS) due to hypoxia, fixation, dehydration, handling, and reconstruction errors are evident. However, changes to the ICS are unknown, leading to four plausible accounts for tissue shrinkage. In all cases, the original morphology can be recovered by scaling the reconstruction by an amount equivalent to the volume of tissue shrinkage (which is 1.0 for D) and then expanding the ECS at the expense of the ICS, or vice versa. A: Water loss from the ECS and the ICS into the solution causes the extracellular and intracellular and total volume to decrease. B: Water loss from the ECS into the solution and no exchange with the ICS causes extracellular volume and total volume to decrease, leaving intracellular volume unchanged. C: Movement of water from the ECS into both the solution and the ICS causes the extracellular and total volume to decrease and the intracellular volume to increase. D: No change in overall tissue volume occurs, as water is simply transferred from the ECS to the ICS. This scenario is incompatible with the 33% volume shrinkage associated with our EM protocol.