The mean is represented by solid lines and the scale on the left axis. The CV is represented by dashed lines and the scale on the right axis. The CV and mean before the artificial variation was normalized to 1. to mean) and mean of YFP fluorescence intensity were plotted against the area of the IM (represented by area ratio of the IM to the original mask), separately. (B) For the generated mismatched masks (MM) and intersected masks (IM), the coefficients of variance (CV) (ratio of s.d. The original masks, shown as white outlines in the middle panel, were artificially shifted 8 pixels in left-to-right direction, generating mismatched masks (right panel). (A) A simplified illustration of mismatched masks generated by artificially shifting a registered live-cell image of YFP emission. The intersections between the mismatched and the original masks were used to mimic masks smaller than the real nuclei (not shown). 300 matched nuclear masks, randomly selected from this pool, were shifted randomly out of their original positions along the horizontal, and (or) vertical, and (or) rotational directions to generate mismatched masks. The identified STAT1-YFP expressing cells from the cell-movement controls were pooled together. The experiment of time-lapse imaging of STAT1-YFP nuclear translocation was repeated three times independently with a cell-movement control ( Materials and method) each time.
of the measured fluorescence of the target protein. Mismatch of nuclear masks with the nuclei increased the s.d. Images with black borders are the registered images the borders indicate the distance by which the original YFP image is shifted away from the fixed-cell images. Arrows point to areas with obvious change in fluorescence intensity. For each image, a magnified view of the field is shown in an inset. Here shows representatives of a registered live-cell image and the fixed-cell image. YFP images of the untreated cells were collected, the cells were then fixed and YFP images of the same field were taken. STAT1-YFP was transiently expressed in Hela cells. STAT1-YFP fluorescence before and after fixation. i = n, n−1,…, 3, 2, represents images collected at i×10 min (r) represents a registered image. I fixed_hoechst was segmented to produce nuclear masks (Step 3), which were applied to process the registered time-lapse images (Step 4).
Nuclear time lapse registration#
I i−1_YFP(r) was then generated after registration with I i_YFP(r), and finally, I 1_YFP(r) was generated (Step 2). With I fixed_YFP as the reference, I i_YFP was registered and generated I i_YFP(r). Immediately after time-lapse imaging, cells were fixed and stained with Hoechst (step 1). (B) Diagram of the automated image analysis.
Flowchart for the time-lapse imaging system.