Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.