Figure 2.

Photobleaching of GFP-labeled mitochondria in CHO cells. (A) Photobleaching with 40 μs bleach time using indicated objectives. Corresponding spot diameters in the focal plane were 0.8 (100×), 1.3 (60×), and 4 μm (20×). (B) Photobleaching as in A. (100× objective) in cells after 6 h fixation in 4% paraformaldehyde. (C) Photobleaching (100× objective) with indicated bleach times. Laser intensity was adjusted to give 20–30% bleach depth. (D) Effect of mitochondrial configuration on GFP diffusion. Cells were incubated with PBS, an hypoosmolar solution (150 mOsm, 1:1 PBS/water), or an hyperosmolar solution (600 mOsm, PBS + 300 mM sucrose) for 5–15 min before bleaching. Each curve is the average of 10–15 fluorescence recovery curves from different cells.