Immersol 518 f immersion media

CLSM fluorescence imaging spectrum was performed with a confocal laser-scanning microscope ZEISS LSM 880 (Carl Zeiss AG, Oberkochen, Germany) equipped with an Airyscan detection unit and a high sensitivity GAsP detector for visible detection. To maximize the resolution enhancement, we used a high numerical aperture (NA) oil immersion objective (Plan-Apochromat 63×/1.4 Oil DIC M27; Zeiss). All imaging was performed using Immersol 518 F immersion media (Carl Zeiss). Laser gain, detector gain, and pixel dwell times were adjusted to maintain the lowest laser power and highest signal to noise ratio in order to avoid saturation and bleaching effects. Three spectral channels were used to record the fluorescence of Laurdan (laser excitation: 405 nm; channel 1: 412–463 nm; channel 2: 472–535 nm) and A5-Alexa 647 (laser excitation: 633 nm; emission: 640–750 nm). CLSM experiments were performed in replicates for more than three times on different samples and days.

Super-resolution imaging was performed in the Neuroscience Light Imaging Facility (NINDS) with a confocal laser scanning microscope Zeiss LSM 880 (Carl Zeiss AG, Oberkochen, Germany) equipped with a 32 channel Airyscan detector (Korobchevskaya et al., 2017 (link)). The whole organ of Corti images were taken in SR mode with a 20X objective (Carl Zeiss). To image the hair cells, we collected a z-stack of images from the stereocilia to the apical half of the hair cell body. We used oil immersion alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective (Carl Zeiss) and Immersol 518F immersion media (n_e = 1.518 (30°), Carl Zeiss). Identical image acquisition settings, no averaging, and optimal parameters for x, y, and z resolution were used in all samples from each independent experiment. Image acquisition and Airyscan image processing were done with Zen Black 2.3 SP1 software (Carl Zeiss) using the Airyscan 3D reconstruction algorithm with the automatic default Wiener filter settings.
Confocal imaging on transfected HEK293 cells was performed in the Microscopy and Imaging Core (NICHD) with an inverted laser scanning microscope Zeiss LSM 780 (Carl Zeiss) equipped with a motorized stage, definite focus and a high sensitivity GaAsp multi-channel spectral detector. We used a 63X/1.4 objective Plan-Apochromat (Carl Zeiss) and the Zen software (Carl Zeiss) for image acquisition.