Plan apochromat 63x 1.4 na oil

COS7 cells co-expressing epitope-tagged receptors labeled fluorescently by anti-tag Fab′ fragments as described above were subjected to FRAP or patch/FRAP experiments as described^{49 (link)}. FRAP studies were conducted at 15 °C, replacing samples after 20 min to minimize internalization. An argon-ion laser beam (Innova 70 C, Coherent, Santa Clara, CA) was focused through a fluorescence microscope (Axioimager.D1; Carl Zeiss MicroImaging, Jena, Germany) to a Gaussian spot of 0.77 ± 0.03 μm (Planapochromat 63x/1.4 NA oil-immersion objective). After a brief measurement at monitoring intensity (528.7 nm, 1 μW), a 5 mW pulse (20 ms) bleached 60–75% of the fluorescence in the illuminated region, and fluorescence recovery was followed by the monitoring beam. Values of D and R_f were extracted from the FRAP curves by nonlinear regression analysis, fitting to a lateral diffusion process^{49 (link)}. Patch/FRAP studies were conducted analogously, except that IgG-mediated cross-linking of epitope-tagged endoglin preceded the measurement^{49 (link)}.

HEK293T cells plated at a density of 80,000–90,000 cells–cm⁻² on a pre-coated PDL 4-well Nunc™ Lab-Tek™ II chamber (Thermo Fisher Scientific, #155382PK) were co-transfected with PylRS/4xtRNA^Pyl (500 ng) and respective tagged TARPs (500 ng) using jetPRIME^® transfection reagent for 24 h in the presence or absence of 250 μM TCO*A. Cells were washed once with cell media to remove excessive TCO*A prior to labeling with 1.5 μM Pyr-Tet-ATTO643 or H-Tet-Cy5 diluted in TCO*A-free medium for 30 min on ice. Subsequently, cells were rinsed three times with ice-cold HBSS and immediately live imaged or fixed for 15 min at RT with 4% FA in PBS followed by three washing steps with HBSS before imaging. Confocal imaging of living or fixed cells was performed using a LSM700 setup (Zeiss) equipped with an oil-immersion objective (Zeiss, Plan-Apochromat 63x/NA 1.4 OIL). eGFP and Pyr-Tet-ATTO643/H-Tet-Cy5 were excited using a 488 nm or 641 nm solid-state laser and respective filter settings. Images were processed in ImageJ (FIJI) adjusting brightness and contrast to identical values for comparison of experiments.

For live imaging, transgenic embryos were mounted in a drop of 1% low melting agarose in filtered artificial seawater on the surface of a 35-mm glass bottom dish (Ibidi μ-Dish) with the eye facing the glass surface.
Confocal images were obtained on a Zeiss LSM780 laser scanning confocal microscope. For live imaging, we used a Zeiss C-Apochromat 40x NA 1.2 water immersion objective, while immunostained samples were imaged using Zeiss Plan-Apochromat 40x NA 1.3 oil, Plan-Apochromat 63x NA 1.4 oil and LD LCI Plan-Apochromat 25x NA 0.8 objectives.
Image data were handled using Fiji [92 (link)]; the Enhance Local Contrast (CLAHE) plugin was used to enhance contrast. The confocal images shown in Figs. 4b, c, 5a, c, and 6a, d were deconvolved using the Fiji plugin DeconvolutionLab2 [93 (link)]; point spread function was calculated theoretically by the PSF generator plugin [94 (link)]. The 3D rendering shown in Fig. 6d was obtained using the ImageJ 3D viewer plugin.
A confocal image stack of an adult brain, immunostained for acetylated tubulin, was used for the 3D reconstruction of the Parhyale optic lobes shown in Fig. 5g. Neuropils were labelled manually using the TrakEM2 Fiji plugin [95 (link)].