N storm inverted microscope

Diatoms were imaged on a Nikon N-STORM inverted microscope with a 100x Oil Immersion TIRF objective with a numerical aperture of 1.49. Four fiber-coupled lasers are available for illumination at 405, 488, 561 and 647 nm, respectively. Corresponding filter sets from Semrock, Chroma and AHF were used to block out all unwanted fluorescence and scattered laser light (see SI Mat/Met for a detailed description). The image was projected onto an Andor Ixon 897 EMCCD camera. To visualize the chloroplasts, the red 647 nm laser was used at low power (1.5 mW end of fiber) for 100 ms. For screening of the FPs, the appropriate excitation wavelength (SI Table S1) was chosen at a power of 40 mW and an exposure time of 100 ms. The photo-conversion test was performed by illuminating the sample with 405 nm light for 1 second at 20 mW. Super-resolution images were recorded at 20 fps with an excitation power of 80 mW. Image stacks of at least 1000 images were used (1000 for Fig. 3A,D and SI Fig. S5, 1200 for Fig. 3E, 2500 for Fig. 3F) to ensure a sufficient localization count. For mEOS3.2 and Dendra2 green excitation is sufficient to localize pre-converted FPs. Dronpa is dark in its ground state, a continuous 405 nm activation of 8 mW in addition to the green excitation was required.

Imaging of NEMO-GFP-expressing cells was performed using an N-STORM inverted microscope (Nikon) in TIRF mode. A 488 nm excitation laser was angled through the back focal plane of an original magnification × 100 TIRF objective (Nikon). A range of laser powers were tested for imaging the samples; as GFP intensity fluctuations are minimal regardless of laser power in the absence of a specialized imaging buffer, similar SOFI-based reconstructions were obtained at all powers tested. As a result, the live-cell movie displayed was obtained at 0.083 kW cm⁻². Emitted signal from the excited GFP molecules was collected by an electron-multiplying charge-coupled device camera (iXon Ultra 897, Andor), with a pixel size of 160 nm.