Reconstruction of a 3D skeleton of the amphibian skeletal t-system. (A) A single confocal section of a mechanically skinned toad iliofibularis fibre with 10 mM Fluo-5N trapped within the t-system. Scale bar: 5 µm. (B) A 3D surface rendered skeleton of the toad t-system reconstructed from a 34×26×7 µm deconvolved confocal image volume illustrating the 3D geometry of tubules. Regions containing a high density of longitudinal tubules (asterisk) were observed. Some of the longitudinal tubules (arrowheads) also appeared to be blind-ended at one end. Scale bar: 4 µm. (C) The magnified view of a 9×5×6 µm sub-volume of the 3D skeleton further illustrates the geometries of the longitudinal tubules and transverse tubules seen at the z-lines. Scale bar: 2 µm. (D) Directionality analysis illustrated by a histogram of the percentage of tubules as a function of the angle relative to the transverse plane shows that a minimum of 2.2% of tubules of the t-system were longitudinal tubules.

Spatial relationship between the t-system and nuclei of mammalian and amphibian skeletal muscle. (A) The longitudinal lateral view of a peripherally located nucleus vitalized with PI (blue) and the skeleton of the t-system (orange) in a mechanically skinned rat EDL fibre. (B) The orthogonal (transverse view) of a 5-µm-deep volume illustrates that the nucleus is closely surrounded by tubules that extend deeper into the fibre. Arrowheads indicate previously reported sub-sarcolemmal tubules that extend near the periphery of the fibre. (C) The longitudinal view of a centrally located nucleus (blue) and the skeleton of the t-system (green) in the toad illustrates the dense tubular mesh wrapping around the nuclei. (D) The orthogonal (transverse) view of a 5-µm-deep volume of the skeleton highlights the cytoplasmic space devoid of tubules, occupied by the nucleus. (E,F) Analysis of the density of tubules as a function of the Euclidean distance from the edge of the region of PI labelling in rat (E) and toad (F) fibres indicate a near-uniform density of tubules in cytoplasmic regions further than ∼0.75 µm away. In both species, an ∼25–50% increase in the tubule densities was observed between 0.2 and 0.7 µm from the edge of the PI staining. Scale bars: 5 µm.

Simulation of the relationship between t-tubule diameter and the convolved image, and estimation of local tubule diameters of the t-systems in living skeletal muscle fibres. (A) A series of simulated images of t-tubules of varying diameters (d). (B,C) The point spread function of the imaging system is elongated in the z-dimension compared to x- and y-dimensions, which accentuates the intensity of tubules whose orientations have a non-zero vertical component (B). This is overcome by convolving the 3D confocal data volumes to effectively achieve an approximate symmetry in x-, y- and z-dimensions (C). Scale bar for B and C: 0.5 µm. (D) A plot showing the normalized mean intensity above background measured along the centre line of the t-tubule of the confocal fluorescence image following the re-blurring (circles). Note that the narrowest detectable t-tubule corresponds to a diameter of ∼40 nm as shown in the magnified region of the plot (inset). (E,F) maximum-intensity projections (2-µm-deep) of 3D t-system skeletons of a rat EDL fibre (E) and a toad iliofibularis fibre (F), colour-coded for the estimated mean local tubular diameters. (G,H) Histograms showing the percentage of tubules as a function of the mean local diameter in rat (G) and toad (H) fibres. The range and mean diameters are similar (rat and toad fibres were 85.4±14.4 nm and 91.2±20.1 nm, respectively; means ± s.d.). Scale bars for E and F: 2 µm.

The effect of osmolarity on the tubule diameters in the rat and toad t-system. (A–C) Histograms showing the distributions of estimated mean tubule diameter of mechanically skinned rat EDL fibres that were incubated in (A) isosmotic (280 mosmol/kg); (B) hypo-osmotic (200 mosmol/kg); (C) hyperosmotic (1000 mosmol/kg) internal solutions are shown on the left. 2-µm-deep projections of the colour-coded maps of the local tubule diameters in the respective osmolarity conditions are shown on the right. (D–F) Histograms for the estimated mean tubule diameter in skinned toad fibres incubated in (D) isotonic 255 mosmol/kg, (E) hypotonic 200 mosmol/kg and (F) hypertonic 1000 mosmol/kg internal solutions for 15 minutes. The colour-coded maps of the local tubule diameters in isotonic, hypertonic and hypertonic conditions, shown on the right, illustrate that changes in local diameter in the transverse tubules were typically larger than those in the longitudinal tubules (arrowheads). Scale bars: 2 µm. Colour scale illustrates estimated mean tubule widths in nm.