LAUSR

Certain morphological parameters of the skeletal muscle tissue can be better understood via 3D considerations. Fluorescent confocal microscopy of thick tissue sections is a well‐established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, thick tissue sections are prone to deformations which may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler‐Poincaré characteristics. To better understand this phenomenon, we studied the horizontal deformation of thick (100 µm) transverse skeletal muscle sections, by comparing the muscle fibre diameters measured on thick sections to muscle fibre diameters measured on thin (10 µm) sections of the same sample. Diameter changes were further correlated with shrinkage in the Z direction (axial shrinkage) and deviation of the muscle fibre preferential axis from the Z‐axis. We showed that the thick sections dilated in horizontal and shrunk in Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in the Z direction. The latter was more pronounced in transversely than obliquely cut tissue sections. The results emphasise that even when shrinkage in the Z direction can be corrected using calibration, it is important to optimise histological protocols to minimise the Z‐axis collapse that could cause horizontal dilation.