Csu x1 m1 confocal scanner

To analyze the kinetics of GEF-H1, wild-type and vimentin-knockout cells were transfected with GFP–GEF-H1 and incubated for 24 h. Confocal images were acquired with a 3I Marianas imaging system (3I Intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss), a Yokogawa CSU-X1 M1 confocal scanner and 63×1.2 NA WC-Apochromat Corr WD=0.28 M27 objective (Zeiss). A heated sample environment (+37°C) and CO₂ control were used. SlideBook 6.0 software (3I Intelligent Imaging Innovations) was used for the image acquirement. Five pre-bleach images were acquired followed by bleaching scans with 100% intensity laser lines over the region of interest. Recovery of fluorescence was monitored 50 times every 200 ms and 300 times every 1 s. The intensity of the bleached area was normalized to a neighboring non-bleached area. Mean scatter plots were calculated from different FRAP experiments and the data were fitted with SigmaPlot 11.0 to f=a×(1×exp(×b×x))+c×(1−exp(−d×x)) double exponential equations. Recovery halftimes were obtained for each recovery curve and the means and standard deviations were calculated.

Cells were transfected with CAV-1-mEGFP and incubated for 24 h. Confocal images were acquired with a 3I Marianas imaging system (3I Intelligent Imaging Innovations, Denver, CO, United States), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss, Oberkochen, Germany), a Yokogawa CSU-X1 M1 confocal scanner and 63 × /1.2 WC-Apochromat Corr WD = 0.28 M27 objective (Zeiss). Heated sample environment (+37°C) and 5% CO₂ control were used. SlideBook 6.0 software (3I Intelligent Imaging Innovations) was used for the image acquirement. Five pre-bleach images were acquired followed by bleaching scans with 100% intensity laser lines over the region of interest. Recovery of fluorescence was monitored 50 times every 200 ms and 300 times every 1 s. The intensity of the bleached area was normalized to a neighboring non-bleached area. Mean scatter plots were calculated from different FRAP experiments and the means and standard deviations were calculated.