Changes in the F.I. of EuTTA nanosheets with HeLa cells before and after treatment with trypsin, PFA, or Triton X-100. (A) Top: Fluorescence images of Calcein in HeLa cells on the EuTTA nanosheet before (left) and after (right) 0.12% trypsin treatment. Bottom: Fluorescence images of the EuTTA nanosheet. See Video 1. (B) Time course of changes in Calcein F.I. in HeLa cells during trypsin treatment. Gray, individual cells; red, average. (C) Same as in B for EuTTA F.I. Gray, individual cells; red, average with cells; blue, average without cells (i.e., average for solution). (D) Same as in C for the corrected F.I. of the EuTTA nanosheet (see Materials and methods for details). Gray, individual cells; red, average. In B, C, and D, trypsin is present during the period shown by the horizontal black line on top of each graph (applied at 90 s). Number of cells, 15 (n = 3). (E) Top: Bright-field images of HeLa cells on the EuTTA nanosheet before (left) and after fixation with 4% PFA (right). Bottom: Fluorescence images of the EuTTA nanosheet. (F and G) Same as in C and D, respectively, but with PFA. In F and G, PFA is present during the period indicated by the horizontal black line on top of each graph (applied at 90 s). Number of cells, 11 (n = 2). (H) Top: Fluorescence images of CellMask in chemically fixed HeLa cells on the EuTTA nanosheet before (left) and after (right) treatment with 0.9% (vol/vol) Triton X-100. Middle: Same as in top for fluorescence images of Calcein. Bottom: Same as in top for fluorescence images of the EuTTA nanosheet. See Video 2. (I) Time course of changes in the F.I. of CellMask in chemically fixed HeLa cells on the EuTTA nanosheet during treatment with Triton X-100. (J) Same as in I for Calcein. (K andL) Same as in C and D but during Triton X-100 treatment. In I–L, the horizontal black line indicates the period of Triton X-100 treatment. Number of cells, 13 (n = 3). The fluorescence images were averaged from 30 frames before (0–58 s) and after treatment (540–598 s).