Diffusion of fluorophores into cardiac myocytes during patch clamp with ∼3 MΩ access resistance (24°C). Each panel shows the micrograph of a myocyte after ∼1 h of diffusion (left), the time course of total fluorescence changes during the experiment (middle), and line scans as indicated in the micrograph, using yellow dots to indicate the starting position of the line scan (right). (A) Sulforhodamine (100 μM) equilibrates with an average time constant of 29 min (middle panel, n = 5). It accumulates eightfold in the cytoplasm (see line scan 2 across the myocyte and into the pipette tip), and it shows a strong longitudinal gradient even after 1 h. Diamonds indicate the starting position of the line scan 2. (B) Carboxyfluorescein (100 μM) equilibrates with an average time constant of 10 min (middle panel, n = 6) and shows longitudinal gradients even after 1 h. Dye accumulation in cytoplasm versus pipette tip remains very small. (C) Asante Na-green (ASG2, 10 μM) also equilibrates with a very long time constant (40 min, middle panel) and shows a longitudinal gradient even after 1 h. Strong fluorescence occurs in myocytes in the absence of Na. (D–F) A 0.5-kD, a 2-kD, and a 10-kD FITC-PEG (100 μM) equilibrate over 1 h with average time constants of 11, 23, and 15 min (n = 6, 5, and 6, respectively), show no evidence of significant binding, and show clear longitudinal gradients even after 1 h. (G) Purified GFP (12 μM) accumulates weakly in the cytoplasm with respect to the pipette tip with an average time constant of 14 min (n = 6). Fluorescence does not show clear longitudinal gradients. AFU, arbitrary fluorescence units.