Actin gfp

To induce the expression of fluorescently tagged proteins chemical transfection or viral transduction were used. In the first case, cells were transfected with Lifeact-GFP using standard chemical transfection reagents (Fugene 6, Promega) and imaged 2 to 3 days after transfection. In the second case, cells were transduced with baculoviruses harboring the constructs Actin-RFP, Actin-GFP, Talin-GFP, and Tubulin-GFP (Life technologies) and imaged 1 to 2 days after transduction. Bright field, phase and fluorescence images were acquired with an inverted epifluorescence fluorescence microscope (AxioObserver.A1, Zeiss) through a 20X air or a 100X oil-immersion objective. Images were captured with a standard charge-coupled device camera (Hamamatsu). For fluorescence measurements, the camera dark noise was subtracted and cells that are high in fluorescence intensity were not included in the analysis. Alignment between fluorescence and AFM images was performed through the alignment of fiducial markers, such as stable cell edges and actin fibers. Data analysis was performed with built-in functions of the software ImageJ.