Figure 2.
Image acquisition and quantitation of rapid collagen deposition mode. (A) (i) 20× original magnification of cells in a 1.9 cm2 well with superimposed original rectangular image acquisition area (red box). (ii) Enlarged red box: corner auto-fluorescence (arrows). (iii and iv) Acquisition area after application of triangular masks to eliminate corner auto-fluorescence. (B) Cytometry and quantitation of the area of deposited collagen I. (i) DAPI-stained nuclei at 20× original magnification in monochrome pseudocolor, scale bar, 1 mm. 600× original magnification (inset). (ii) Red scored nuclei by Count Nuclei module for cytometry. (iii) Immunostained deposited collagen I. (iv) Regions with fluorescent pixel intensity above a selected value based on controls are demarcated by the software in green for quantitation of deposited collagen I area at 100× original magnification. (C) Shading correction for non-homogenous fluorescence during image acquisition for unbiased quantitation at 20× original magnification. (i) Non-homogenous fluorescence background. (ii) Fibronectin immunofluorescence acquired with uneven fluorescence. (iii) Same image after background fluorescence removal. (D) Densitometry versus image analysis for collagen I (Col I) quantitation. Optically, fluorescence intensity (FIT) and area were similar, correlating well with densitometry. TGFβ1-induced increase of collagen I deposition was detected in similar amount and tendency optically and biochemically. Data are expressed as mean ± SD, calculated from triplicates and as a relative percentage of all treatments. Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindoldilactate; DxS, dextran sulphate (rapid deposition mode); TGFβ1, transforming growth factor-β1.