ISI imprinting and FSM on the mitotic spindle. (a) Photoconversion ISI mode in a human transformed cell line (U2OS) and photobleaching ISI mode in a Drosophila S2 cell. Bar, 5 µm. (b) Orthogonal views of a metaphase Drosophila S2 cell before and after ISI show the 3D character of the imprinted speckles. Bar, 5 µm. (c) An FSM and an ISI-prepared metaphase cell. ISI cells (n_ISI = 20) displayed an approximately sixfold increase in speckle amplitude compared with FSM cells (n_FSM = 20, chosen and imaged by an independent experimenter) generated by expression of leaky GFP-tubulin levels in the absence of the promotor. Error bars represent SD. ADU, analog-digital units. Bar, 5 µm. (d) Post- to pre-ISI fluorescence signal ratio image in pseudocolor in a Drosophila S2 cell. ISI-induced fluorescence fluctuations, but not mean levels, are dependent on the dynamics of the structure to which the fluorophore is bound. Cytoplasmic pool dynamics induces a fast wash out of the speckle pattern already at the first post-ISI frame (red box). Bar, 2 µm.