Plan apo tirf objective

The cells were studied with bright-field and fluorescence microscopy using a Nikon ECLIPSE TE2000-E microscope (Plan Apo TIRF objective, magnification 60×, NA = 1.45). Using the bright-field technique, the images were captured with a digital camera (DS-2M BW; Nikon, Japan). For confocal microscopy, the Nikon C1 system was used (light source: xenon–argon laser 488, excitation filter: EX510-560, absorption filter: BA590). The images were recorded within the same field of view for 60 min at a rate of 1 frame/s in the bright-field mode and at a rate from 1 frame/s to 1 frame/10 min in the fluorescence microscopy.
The hypotonic solutions were obtained by either (i) the addition of distilled water (J. T. Baker, Poland) to the Leibovitz’s L-15 medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS) (Leibovitz’s medium) or (ii) the preparation of different sucrose-water solutions (Sigma, United States). In the first case, the Leibovitz’s medium was diluted by 90, 80, 60, 40, or 20% with distilled water, leading to Leibovitz-water solutions with osmolarities equal to 32, 63, 126, 189, and 252 mosM/L. In the second case, different sucrose-water solutions whose osmolarities corresponded to those of the Leibovitz-water solutions were prepared. Finally, the cells were also measured in the distilled water.

All TIRF images except for Fig. S5 C were acquired with the DeltaVision OMX SR (GE Healthcare) equipped with three PCO 15-bit CMOS cameras with a 60×/1.42 NA Plan Apo objective (Olympus) with 1.516 or 1.518 refractive index oil (Cargille) in ring-TIRF mode with either sequential or simultaneous excitation. Images were acquired with Acquire SR and deconvolved (default settings) via softWoRx. TIRF images in Fig. S5 C were acquired on a Nikon Eclipse Ti microscope equipped with a Borealis beam-condition unit (Andor Technology), a 60×/1.49 NA Plan Apo TIRF objective (Nikon), and an iXon Ultra EMCCD camera. Environmental control (37°C/5% CO₂; Okolab) was used. Acquisition was controlled with Micro-Manager (UCSF; Edelstein et al., 2014 (link)).