Fig. 4.

Identification of metabolic states of germ cells during differentiation. Fluorescence intensity image of a C. elegans germ line (A) excited at 740 nm and (B) excited at 880 nm. Histone-GFP fusion protein identifies the position and differentiation state of the germ cells that are indicated with different colors: distal mitotic region (blue), proximal mitotic region (red), transition zone (green), and meiotic pachytene (cyan). A red cursor of 5-μm diameter selects the region of interest of a germ cell in the intensity image at (C) 880 nm and (D) 740 nm. (E) Phasor plot of the FLIM image excited at 740 nm (color scale defined in Fig. 1D). (F) Scatter plot of the cell phasor of all germ cells excited at 740 nm. Every cell phasor (squares) is represented with a color that corresponds to its differentiation state in B. The distribution of distal mitotic cells in blue (blue, n = 14), proximal mitotic region (red, n = 20), transition zone cells (green, n = 83) are clearly separated. The mean values of clusters are represented by the colored stars and the SD by the dotted lines. (G) Scatter plot of the mean values of cell phasor distributions in distal mitotic region (blue), proximal mitotic region (red), and transition zone (green) for n = 6 independent C. elegans germ line. Independent samples are represented with different symbols. (H) Scatter plot of the mean values of the cell phasor distributions for n = 6 independent germ lines. Each sample is translated in the phasor plot as to make all of the distal mitotic region values coincident. The SDs of the proximal mitotic region and transition zone are represented by the dotted lines. (I) Zoomed image of the mitotic region of the C. elegans germ line excited at 880 nm in B. Cells are numbered in a distal to proximal direction. Blue cells belong to the distal mitotic region and red cells belong to the proximal mitotic region. (J) Scatter plot of the phasor average values of the 20 germ cells indicated in I.