Fig. 3.

CLEM analysis of GFP-positive and GFP-negative cells derived from TH-GFP iPSCs. a Diagram representing the comparative analysis of GFP-positive and -negative cells in CLEM analysis (Figs. 4 and 7) and live cell imaging (Fig. 5, Additional file 3: Figure S2). Neurons expressing high levels of GFP (green in the diagram) were defined as “GFP-positive” cells. Cells expressing no levels of GFP and with a neuronal shape, which were probably other neuronal subtypes or neural progenitors (white in the diagram), were defined as “GFP-negative” cells. Neurons expressing low levels of GFP (light green in the diagram) were not used in either CLEM analysis or live cell imaging. b Scheme of the CLEM procedure. Neurospheres differentiated from TH-GFP iPSCs were dissociated and reseeded on the gridded coverslips. After 7–10 days of culture, the differentiated neurons consisted of a mixture of GFP-positive (green) dopaminergic neurons and GFP-negative (white) non-dopaminergic neurons. Brightfield and fluorescent images were taken of cells on the gridded coverslips, and the cells were further fixed and flat-embedded for electron microscopic analysis. The cells of interest in the ultrathin sections were identified with help from the grid patterns and cell shapes using an SEM. c The GFP (right) and merged (brightfield + GFP, left) images of TH-GFP iPSC-derived cells on the gridded coverslips. The white box indicates the origin of the enlarged image (c, left). Scale bar, 100 µm. d The brightfield and GFP (left), GFP and SEM (center), and SEM (right) images. Asterisks and arrows indicate GFP-positive and -negative cells, respectively. Scale bar, 20 µm