Fig. 2.

Images and image analysis of fluorescent drug accumulation in HNC monolayers. (A, B) Images of Cal33 monolayers stained with Hoechst alone, or Hoechst plus ellipticine, idarubicin, daunorubicin, or doxorubicin. Cal33 cells were seeded into 384-well monolayer assay plates at 1,000 cells per well and cultured for 24 h before 10 μM of the indicated compounds was added to wells that were incubated for an additional 15 min before fixation in formaldehyde containing Hoechst for 30 min. After washing with PBS, images of two fields of view of 2D Cal33 monolayers were acquired sequentially on the IXM by using a 10 × objective in each of three fluorescent channels: DAPI, FITC, and TRITC. Representative grayscale images from numerous independent experiments of each of the fluorescent channels are presented along with the corresponding color composite images. (C) MWCS image analysis module nuclear and whole cell masks. The MWCS module image segmentation identified the Hoechst 33342 stained Cal33 nuclei and created nuclear masks for each cell. After applying user-defined background average intensity thresholds, typically 200 in both Ch2 (FITC) and Ch3 (TRITC), the MWCS module image segmentation created total cell masks for each cell. The nuclear mask was used to quantify the mean integrated fluorescence intensity of Hoechst within Cal33 nuclei, and to count the number of cells. The derived Cal33 whole cell masks in Ch2 and Ch3 were then used to quantify the mean integrated fluorescence intensity of the drugs in the FITC and TRITC channels, respectively. Ch2, fluorescent channel 2; Ch3, fluorescent channel 3; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; MWCS, multi-wavelength cell scoring; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine.