Fig. 1.
TIRF imaging of actin networks and their reorganization in the cortex of Dictyostelium cells. (A) Illumination at a high incidence angle (blue) generates an evanescent field that illuminates the cortical actin network of 100–200-nm thickness (green). Molecules diffusing in the cytoplasm are illuminated only for the fraction of time they persist within the evanescent field, a period that is prolonged by incorporation into the cytoskeleton (red double arrows). (B) TIRF image of the actin network visualized by LimEΔcoil-GFP in a cell immobilized by tight compression under an agar sheet, taken at an exposure time of 100 msec. (C) Two-dimensional color space where It indicates color coding of the gray scale for the first frame (green) and It + 1 indicates color coding of the gray scale for the second frame (red). Mixed colors (yellow tones) represent structures that persist from the first frame to the second frame with constant or slightly varying fluorescence intensity. (D) Superposition of two TIRF images of the cell shown in B, recorded at a 500-msec interval and color-coded as in C. Areas of very densely packed actin filaments with fluorescence intensities beyond the linear range of the 0–255 gray scale are left uncolored. (E–G) gainfi in a TIRF image series recorded at 100-msec intervals. (E) Snapshot at the beginning of the series. (F) gainfi during a period of 200 msec obtained by image subtraction, colored in red. (G) gainfi obtained by subtracting 20 consecutive frames from each other, presented as a maximum projection over the period of 2 sec. The gray and color bars indicate contrast enhancement. (Scale bars: 5 μm.)