Figure 1
Epifluorescence–based quantification of cytoskeletal organization, nuclear shape and chromatin condensation. All panels depict the same example cell/nucleus. (A) Overlay of fluorescence images for TRITC (phalloidin) and DAPI channels obtained on an epifluorescence microscope using a 20× objective. Quantification of F-actin fibre fluorescence intensity (B) and fiber orientation (C) obtained from the raw images. Fluorescence intensity of the nucleus before (D) and after (E) band-pass filtering. Fluorescent speckles resulting from areas of high chromatin condensation are clearly visible in the zoomed-in image shown as an inset. (F) Averaged fluorescence intensity profile as a function of radial distance I(r). Black squares correspond to fluorescence intensities recorded, and the imaged nucleus is taller than the depth of focus of the objective lens. Red line corresponds to the ellipse obtained when fitting the fluorescence intensity profile of the outermost pixels. Left axis shows the fluorescence intensity values from the analysed image, while right axis shows the height profile estimated using the calibration factor. (G) x-z reconstruction of the nucleus as obtained from a confocal image stack. Overlaid is the estimated gross nuclear morphology as obtained from the fit shown in (F). Scale bar is 50 µm in panels A–E and 5 µm in panel G. In panels B–E a false colour scale has been used to improve visualization. Inset in (C) exemplifies the computed orientation of the nucleus (θnuc) and the average orientation of the fibres (θfib).
