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Pipeline publication

[…] and going through a 100-m optical fibre to enlarge pulse width (100 ps) used for depletion. A doughnut-shaped laser beam was achieved through two lambda plates. Fluorescence light between 650 and 740 nm was collected using a photomultiplier, using a HCX PL-APO CS × 100/1.40 NA oil objective and a pinhole open to one time the Airy disk (60 μm). A fivefold zoomed area of 512 × 512 pixels, corresponding to a pixel size of 30 nm, was scanned at 50 Hz. In-depth acquisitions were performed by scanning hippocampal slices with an increment of 1 μm in z over 60 μm. When indicated, raw data were deconvoluted using the measured point spread function of the system and the Richardson–Lucy algorithm with Huygens Professional (Huygens Software). 3D reconstructed movies were made using Imaris (Bitplane)., Neurons electroporated with Homer1c-GFP, BirAER and AP-Nlg1 or AP-LRRTM2 and cultured for 2 weeks were labelled with high concentration of mSA-Atto594 (100 nM) for 10 min, and then rinsed, mounted in Tyrode solution and observed under the same set-up used for uPAINT. The laser bench (comprising 488, 561 and 642 nm lasers, 100 mW each, Roper Scientific) has a second optical fibre output connected to an illumination device containing galvanometric scanning mirrors (ILAS, Roper Instrument) steered by MetaMorph. It allows precise spatial and temporal control of the focused laser beam at any user-selected region of interest within the sample for targeted photobleaching. Switching between the two fibres for alternating between imaging and bleaching is performed in the millisecond range using a mirror. Oblique illumination acquisition was performed using the 561-nm laser at low power (300 μW at the front of the objective) to precisely image molecules accumulated at the substrate level. After acquiring a 10-s baseline at 0.5–1 Hz frame rate, rapid selective photobleaching of two to three synapses was achieved at higher laser power (3 mW at the front of the objective) during 200–300 ms. Fluore […]

Pipeline specifications

Software tools Huygens, Imaris, MetaMorph