Computational protocol: Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

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[…] Larvae were dissected in ice-cold HL-3, fixed for 10 min using 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) and blocked for 30 min in PBT (PBS with 0.05% Triton X-100, Sigma) containing 5% normal goat serum (Jackson ImmunoResearch). Preparations were incubated with primary antibodies at 4 °C overnight. After one short and three × 20 min washing steps, the filets were incubated with secondary antibodies for 2 h followed by another three washing steps. The samples were mounted in Vectashield (Vector Laboratories) for confocal imaging or kept in PBT for dSTORM measurements. Primary antibodies were used in the following dilutions: monoclonal antibody (mAb) BrpNc82 (1:250, provided by E. Buchner) and rabbit-GluRIID (1:1,000, provided by S.J. Sigrist). Alexa Fluor 488-conjugated mouse (Invitrogen) and Cy3-conjugated rabbit (Dianova) antibodies were used at 1:250. Images were acquired with a Zeiss LSM5 Pascal confocal system (objective: 63 × , numerical aperture 1.25, oil). For each set of experiments, all genotypes were stained in the same vial and imaged in one session. To estimate synapse numbers laser power was adjusted individually for each NMJ.Brp punctae and GluRIID clusters (NMJ 6/7, segments A2, A3) were examined using ImageJ software (National Institutes of Health) in principle as previously described. After background subtraction, a Gaussian blur (0.9 px s.d.) was applied to maximum z-projections of confocal stacks and masks were generated (threshold mean grey value of 25 for Brp and 30 for GluRIID). After superimposing the binary mask on the original blurred image, spot detection and segmentation via the ‘Find Maxima’ operation was performed to extract particle numbers.To estimate the number of release sites (N) per AZ, the modelling prediction of N (average value of both models) was divided by the number of AZs on muscle 6 identified in confocal images, that is, half of NMJ 6/7 (ref. ). [...] The mAb BrpNc82 was used at a dilution of 1/2,000 to identify AZs. Goat anti mouse F(ab′)2 fragments (A10534, Invitrogen) were labelled with Cy5-NHS (PA15101, GE Healthcare) according to standard coupling protocols given by the supplier. Purification of the conjugates was performed by use of gel filtration columns (Sephadex G-25, GE Healthcare). The degree of labelling was determined by absorption spectroscopy (Jasco) as 1.3 for studies of the CAZ ultrastructure and 1.3–1.5 for dilution experiments. Samples were stored in 0.2% sodium azide in PBS and for the experiments, Cy5-labelled secondary antibody was used at a concentration of 5.2 × 10−8 M.For dSTORM imaging with Cy5, the sample was embedded in photoswitching buffer, that is, 100 mM mercaptoethylamine, pH 8.0, enzymatic oxygen scavenger system (5% (wt/vol) glucose, 5 U ml−1 glucose oxidase and 100 U ml−1 catalase) and mounted on an inverted microscope (Olympus IX-71) equipped with an oil-immersion objective (60 × , numerical aperture 1.45, Olympus) and a nosepiece stage (IX2-NPS, Olympus). For excitation of Cy5, a 641-nm diode laser (Cube 640–100C, Coherent) was used. Telescope lenses and mirror were arranged on a translation stage to allow for switching between wide-field, low-angle/highly inclined thin illumination and total internal reflection fluorescence imaging.Fluorescence light from Cy5 was filtered by a dichroic mirror (650, Semrock) and a band- and long-pass filter (BrightLine 697/75, RazorEdge 647, Semrock), and imaged on an electron-multiplying CCD camera (EMCCD; Ixon DU897, Andor Technology). Additional lenses were used to generate a final camera pixel size of 107 nm. Fifteen thousand frames were recorded with a frame rate of 100 Hz at an irradiation intensity of ~5 kW cm−2. For imaging A488, a 488-nm laser (Sapphire 488 LP, Coherent) and a polychromatic dichroic mirror (410/504/582/669, Semrock) were used. Fluorescence light from A488 was reflected by a dichroic mirror (630 DCXR, Chroma) and imaged on a second EMCCD camera equipped with a band-pass filter (HQ535/50, Chroma).Goat anti mouse IgG labelled with A532 (A11002, Invitrogen) and A700 (A21036, Invitrogen) was used at a concentration of 6.25 × 10−9 M. The degree of labelling was determined as 2.0 (A700) and 4.5 (A532). Imaging of A532 and A700 by dSTORM was performed in PBS containing 100 mM mercaptoethylamine, pH 8.3. Using appropriate filter sets (dichroic mirrors: 650 or 545/650; band-pass filters: RazorEdge 647 or BrightLine 582/75, Semrock), the samples were irradiated at 641 nm (A700) or 532 nm (NANO 250-532-100, Linos; A532) at ~5 kW cm−2. For titrations of A532-labelled secondary antibodies (), fluorescence light from A700 and A532 was separated by a dichroic mirror (630 DCXR, Chroma) and imaged on two EMCCD cameras.Super-resolution images were reconstructed using the software package rapidSTORM. Only fluorescence spots containing >1,000 photons were analysed. Double-spot emission was analysed by a two-kernel analysis as described applying a maximum two-kernel improvement of 0.1. Raw localization data obtained from rapidSTORM was examined and further processed with ImageJ. A subpixel binning of 10 nm px−1 was applied. Representative images in and magnified views in are shown with 7 nm binning for clarity.To measure CAZ (defined by BrpNc82) area and localization numbers, masks were created by applying a Gaussian blur (1 px s.d.) followed by a minimal threshold (0.15 counts). After a minimum overlay of the original data with the masks, CAZs were then identified via their area (300 px to infinity). For the comparison of genotypes, a total of 812 CAZs in controls, 776 in brpnude and 257 in rab3rup were analysed and data (presented as mean±s.e.m.) were acquired in two imaging sessions, each containing all three genotypes stained in the same vial. Images with a background of >2.3 single spots per μm2 were excluded from the comparative analysis. Unspecific background labels exhibited equal localization counts in all genotypes (average counts control: 12.0±0.2 localizations s.e.m., n=16 NMJs; brpnude: 12.0±0.1, n=13; rab3rup: 12.4±0.3, n=11), indicating comparable imaging settings.For the investigation of different motorneurons, a total of 963 (type Ib) and 579 (type Is) CAZs (from NMJ 6/7, segments A2 and A3) were analysed to determine the gradient. Double-stainings included horseradish peroxidase directly conjugated to A488 (1:250, Jackson ImmunoResearch) for visualization of boutons. In the representative images, the epifluorescence signals were background subtracted and normalized.To specify the localization precision of dSTORM images, localizations of unspecific background label (n=21,436) from all three genotypes were analysed using ImageJ. Masks were created as described above (Gaussian blur with 1 px s.d., threshold 0.08 counts). Within a dSTORM image, only selections with a size between 16 and 100 px (10 nm px−1) and an ellipticity ≥0.95 were anaylzed. The coordinates of localizations within a single selection were aligned to their centre of mass and a 2D histogram of all localizations (209,537 in total) was generated (binning: 4 nm × 4 nm). A 2D Gaussian function was fitted to this histogram (adjusted R2=0.995). The s.d. of the Gaussian function (σx,y=(σx+σy)/2)) was determined as 7.16±0.02 nm and is stated as localization precision in this work (). This value is comparable to the localization precision obtained with an alternative method based on nearest neighbour analysis ().For the investigation of CAZ-units, those structures were chosen that were not grouped together and were viewed en face, that is, with the AZ membrane perpendicular to the optical axis. For the manual selection of CAZ-units, the genotypes were blinded.To calculate the radial distribution (), Mathematica 9.0 (Wolfram Research) was used to automatically calculate the centre of each chosen CAZ-unit as the centre of mass (that is, the average localization of all pixels of the CAZ-unit weighted with the pixel value). Subsequently, the distance of each pixel to the centre of mass was calculated. These distances were then binned, the pixel values were added to the corresponding bins and the values were normalized by the area of each radial slice. The resulting distributions were averaged across all chosen CAZ-units, resulting in mean and s.e.m. values for the radial distributions of each genotype. […]

Pipeline specifications

Software tools ImageJ, rapidSTORM
Applications Super-resolution imaging, Microscopic phenotype analysis
Organisms Drosophila melanogaster