Fig. 3.
Probing nuclear and cytoplasmic diffusive tortuosity. (A) Fluorescence recovery after photobleaching (FRAP) protocol for adult ventricular myocyte AM-loaded with calcein (the FRAP marker) and Hoechst 33342 (to identify nuclear regions). Superfusion with 0Na0Ca solution to block contraction. Bleaching in 3 × 3 μm region of cytoplasm evokes cytoplasmic calcein diffusion, quantified in terms of rate constant from exponential best-fit (gray dotted curve). (B). FRAP experiments performed on adult ventricular myocytes with bleaching regions set to the outline of nuclei or a 3 × 3 μm region within nuclei (identified by Hoechst 33342 fluorescence). FRAP repeated in bulk cytoplasm using the same bleaching configuration (n = 10–15 cells/histogram). Emptying sarcoplasmic reticulum Ca2 + stores with thapsigargin (Thapsi; 10 μM) did not affect calcein diffusivity. Experiments performed on neonatal ventricular myocytes in 0Na0Ca solution or normal Tyrode (NT) (n = 20–25 cells/histogram).