Alpha plan apochromat 100 1.46 oil

Cells were spotted on a 2% agarose pad made with 0.22 µm-filtered M2G. Imaging was performed at room temperature. All images were acquired on either an N-STORM microscope (Nikon) or a custom-built setup assembled on a commercial Axio Observer D1 microscope stand (Carl Zeiss). The N-STORM microscope was equipped with a CFI Apo TIRF 100 × oil immersion objective (NA 1.49), lasers emitting at 405 nm and 561 nm (Agilent Technologies, Santa Clara, CA) and a built-in Perfect Focus system. Raw single molecule data were taken in a field of view of ∼43 × 43 µm² (256 × 256 pixels) with an Andor iXon X3 DU 897 EM-CCD camera (Andor Technology, South Windsor, CT) at a frame rate of 28.5–29 frames per second (fps). Under this acquisition condition, diffusing molecules appeared blurred and were rejected from our analysis. The custom-built microscope set up (Huang et al., 2013 (link)) was equipped with a 100 × /1.46-NA oil-immersion objective (alpha Plan-Apochromat 100 × /1.46 oil, Carl Zeiss), lasers emitting at 405 nm (50 mW, CrystaLaser, Reno, Nevada) and 568 nm (Innova 300, ∼400 mW, Coherent, Santa Barbara, CA). Fluorescence was recorded with an ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics) at a frame rate of 100 fps and a field of view of 23 × 23 μm².

Samples were examined using a Leica TCS SP5 laser scanning microscope (Leica Biosystems) with an HCX PL APO lambda blue 63 × /1.4 oil immersion objective or a VisiScope CSU-W1 spinning disk confocal microscope (Visitron Systems GmbH, Pulcheim, Germany) with a PlanApo N 60 × /1.42 oil immersion objective (both microscopes belonging to the Cellular Imaging Laboratory of the Biological Research Centre, Szeged, Hungary). Certain confocal and superresolution images were captured with a STEDYCON (Abberior Instruments, Göttingen, Germany) STED (stimulated emission depletion) superresolution system attached to an Axio Observer Z1 inverted epifluorescence microscope (Zeiss, Oberkochen, Germany) equipped with an alpha Plan-Apochromat 100 × /1.46 oil immersion objective. Multiple images were taken from approx. 5 µm depth of field in case of human cells and sections or 20 µm in case of mouse sections. These images were then merged into z-projections in FIJI. In some cases, the percentage of colocalization was provided to simplify the visualization of certain staining.