Comparison of actin speckle microscopy with phalloidin staining. Primary cultures of newt lung epithelial cells were microinjected with X-rhodamine actin. Time-lapse FSM was performed on a spinning disk confocal microscope system. Light from a 50-mW Krypton-Argon ion laser (Melles Griot, OmniChrome) was delivered by a single-mode fiber optic (Point Source) to a Yokogawa spinning disk confocal scan-head (Ultra-View; PerkinElmer) on an inverted microscope (TE300 Quantum; Nikon). Excitation wavelength was selected by a filter-wheel apparatus (Sutter Instruments Co.) containing excitation filters for 488 and 568 nm (Chroma). Emission was selected by multiple bandpass dichromatic mirror and emission filter (Chroma). Images were collected by a 100x 1.4NA Plan-Apo DIC objective lens (Nikon) and captured with an Orca 2 camera (Hamamatsu). Images were collected in the ventral focal plane at 10s intervals. Following live cell imaging, cells were fixed with paraformaldehyde, permeabilized and stained with Alexa 488 phalloidin. Images were processed as follows: (1) background subtraction; (2) 3 x3 low pass filter; (3) unsharp mask filter. Video corresponds to Fig 1 and video 1 in J Cell Biol, 158:31-37, 2002.
|Spatial Axis||Image Size||Pixel Size|
|Time||10 seconds||8.3 min|