4 channel easy3d superresolution sted optics module

Two-color STED micrographs were acquired using a 4-channel easy3D superresolution STED optics module (Abberior Instruments, Goettingen, Germany) coupled with an Olympus IX83 confocal microscope (Olympus, Tokyo, Japan) and equipped with an UPlanSApo 100x (1.4 NA) objective (Olympus, Tokyo, Japan) (available in the LIMES imaging facility). Two-color STED-microscopy was realized by sequential imaging of the two channels, using pulsed 561 nm and 640 nm lasers for the excitation of Alexa 594 and 647, respectively. For depletion, a pulsed 775 nm STED laser was used. Signals emitted from Alexa Fluor 594 and Alexa 647 dyes were detected using 580–630 nm and 650–720 filters, respectively. Depending on the experiment, pixel size was set to 15 nm for membrane sheets and 25 nm for cells. For all images, pinhole size was set to 60 µm. For the imaging of membrane sheets, the focal plane was adjusted slightly above and below the membrane sheet in order to check its two-dimensional structure briefly before imaging. This is possible because membrane sheets become immediately unfocussed when the focal plane is displaced in the z-axis, whereas cells or incompletely generated membrane sheets still show structural elements (Figure 4—figure supplement 1).

For STED and confocal microscopy, a 4-channel easy3D super-resolution STED optics module (Abberior Instruments) coupled with an Olympus IX83 confocal microscope (Olympus, Tokyo, Japan) and equipped to an UPlanSApo 100 × (1.4 NA) objective (Olympus, Tokyo, Japan) was used. Atto488 and Alexa488 were excited with a 485 nm laser and recorded with combined 500–520 nm and 532–558 nm filters. Alexa594 was excited with a 561 nm laser and recorded with a 580–630 nm filter. Atto647N, STAR RED and iFluor647 were excited with a 640 nm laser and detected with a 650–720 nm filter. The pinhole size was set to 60 µm. For STED microscopy, pulsed STED lasers 595 nm (for Alexa488 and Atto488) and 775 nm (for Alexa594, Atto647N, and STAR RED) were used for depletion. STED images were recorded via a time-gated detection with 0.75 ns delay and 8 ns gate width. Depending on the experiment, pixel size was set to 20–40 nm.
For intracellular vesicles visualized with the L1–7-antibody (Fig. 1B), the cell body was recorded in the focal plane where most vesicles were visible. In all other experiments, the focal plane was adjusted to the basal membrane area.