Csu w1 confocal unit

Trypsinized cells were transferred onto μ-Slide 8-well (Ibidi, Martinsried, Germany) coated with Laminin-511 and cultured for 1 h at 37°C and 5% CO₂. Then, cells were fixed and subjected to smFISH as described above using Nanog exonic probes. After the image acquisition, cells were subjected to immunostaining, as described in the immunostaining section above, using the anti-Nanog antibody and Alexa Fluor 647 goat anti-rat IgG (H+L) (1:500, Life Technologies). The images were acquired using an Olympus IX83 microscope with a CSU-W1 confocal unit, 60× Olympus oil-immersion objective of 1.42 NA; 101 z planes per site, spanning 15 μm, were analyzed. smFISH images were filtered using ImageJ, with a one-pixel diameter 3D median filter, and subjected to background subtraction via a rolling ball radius of 2 pixels. Further fluorescence subtraction (100, 16-bit pixel unit) for almost complete removal of background intensity and maximum-intensity projection was subsequently performed. Immunostained images were filtered with a two-pixel diameter 3D median filter, and subjected to fluorescence subtraction (300, 16-bit pixel unit) for almost complete removal of background intensity and maximum-intensity projection, using ImageJ. The freehand selection tool of ImageJ was used for measurements of integrated signal intensity in each cell.

Trypsinized cells were transferred onto μ-Slide 8-well coated with Laminin-511 and cultured overnight at 37°C and 5% CO₂. The next day, after the medium was changed, cells were subjected to live imaging by an Olympus IX83 microscope with a CSU-W1 confocal unit and a 60× Olympus oil-immersion objective of 1.42 NA for quantification of transcriptional dynamics. Fluorescence images were captured using an iXon3 EMCCD camera, equipped with a 488-nm laser, a stage-top microscope incubator (5% CO₂ at 37°C; Tokai Hit, Shizuoka, Japan), and an ASI MS-2000 piezo stage (ASI, Lyon, France), and were analyzed using Metamorph software; 41 z planes per site, spanning 12 μm, were acquired with a 2-min interval time for 4 h. Acquired images were filtered with a one-pixel diameter 3D median filter using ImageJ. Detection of fluorescent spots was performed using the “Spot” function in Imaris with a spot diameter set at 0.75 µm (semi-automatic detection). For observation of individual mRNA spots, a 100× Olympus oil-immersion objective of 1.40 NA was used. See Supplementary Video S3 and Figure S3 for details.