Uplansapo 100 1.4na objective

For the confocal imaging experiments, hippocampal neurons were plated on glass coverslips. Following the same protocol as for the samples imaged with NanoSIMS, the neurons were first fixed and then immunostained. For comparison with the NanoSIMS results observed from the anti-Vglut1 gold-conjugated nanobody, we incubated the neurons with a FluoTag-X2 anti-vGlut1 nanobody directly conjugated to STAR580 (NanoTag, N1602), for 1 h at RT, in blocking solution. Likewise, to validate the results obtained with SIMS using the anti-mouse gold-conjugated secondary nanobody, we incubated the hippocampal neurons with the same mouse anti-TOM20 primary antibody, and with FluoTag-X2 anti-mouse secondary nanobodies conjugated to STAR635P (NanoTag, N2002), for 1 h at RT, in blocking solution. Following the incubations, the neurons were washed with PBS and then embedded in Mowiol. Confocal imaging was performed on an Abberior QUAD scan STED/confocal microscope (Abberior GmbH, Göttingen, Germany) equipped with a UPlanSApo 100 × 1.4 NA objective (Olympus Corporation, Shinjuku, Tokyo, Japan) and an EMCCD iXon Ultra camera (Andor, Belfast, Northern Ireland, UK). The samples were excited using pulsed 485 nm, 580 nm, and 640 nm lasers for imaging the autofluorescence background signal and the proteins of interest, vGlut1 and TOM20, respectively. The pinhole size was set to 1 airy unit.

A 4-channel easy3D super-resolution STED optics module (Abberior Instruments, Göttingen, Germany) coupled with an Olympus IX83 confocal microscope (Olympus, Tokyo, Japan) and equipped with an UPlanSApo 100 × (1.4 NA) objective (Olympus) was used for confocal and STED microscopy (available in the LIMES imaging facility). Atto488 and Alexa488 were excited with a 485 nm laser and recorded with combined 500–520 nm and 532–558 nm filters. Atto594 and Alexa594 were excited with a 561 nm laser and recorded with a 580–630 nm filter. iFluor647 was 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 775 nm for Alexa594 and iFluor647) were used for depletion. STED images were recorded employing a time-gated detection with 0.75 ns delay and 8 ns gate width. Depending on the experiment, pixel size was set to 20–50 nm. Z-Stacks were acquired by recording images every 400 nm starting from the basal membrane of the cell and from then on moving up to 1600 nm above the basal membrane.