Journal: STAR Protocols

Article Title: Protocol for matching protein localization to synapse morphology in primary rat neurons by correlative super-resolution microscopy

doi: 10.1016/j.xpro.2024.103160

Figure Lengend Snippet: Correlation between PALM and STED images (A) Merged image showing the localization of the Tetraspeck beads obtained from PALM and confocal channels (red and yellow, respectively). The centroids of each bead were determined with PALMTracer, imported in Napari, and represented here as circles. The lines show the connections made between the beads in the two channels, which served us to establish a transformation matrix converting the object localizations in PALM onto the confocal image. (B) Using this correspondence, the coordinates of the beads in PALM were transformed so as to align to the beads in the confocal image (now in magenta). The white grids display the local image deformations in the process. (C) Zoom on a dendritic area from Figure 3 F showing the reconstructed PALM images of the Xph20-mEos3.2 signal before (red) and after (magenta) transformation. (D) Superimposition of the STED image of GFP-actin amplified with Atto647N conjugated antibody, Ab-Atto647N (white), with the transformed PALM image of PSD-95 labeled with the Xph20-mEos3.2 intrabody (magenta). (E) Image of a neuron expressing GFP-actin amplified with Atto647N and imaged with STED. (F) Correlative image between mEos3.2-actin (PALM) and GFP-actin signals (STED) of the dendritic segment delineated in (E). (G) Representative images of individual dendritic spines containing 0, 1, 2, or 3 PSD-95 nanodomains (appearing in white). The spine contour is delineated in red and the corresponding projected area is indicated. (H) Histogram showing the proportion of spines having 0, 1, 2, 3, or 4 nanodomains. Data are represented as mean ± SEM and dots represent individual neurons ( n = 7). (I) Bar graph showing the positive correlation between the number of PSD-95 nanodomains per spine, and the projected area of the whole spine. Data are represented as mean ± SEM and dots represent individual spines ( n = 105 spines from 7 neurons).

Article Snippet: The image stacks obtained in PALM were analyzed using the PALMTracer software, a MetaMorph add-on., , Single molecule localization was achieved using wavelet segmentation, then filtered out based on the quality of a 2D Gaussian fit.

Techniques: Transformation Assay, Amplification, Labeling, Expressing

Journal: STAR Protocols

Article Title: Protocol for matching protein localization to synapse morphology in primary rat neurons by correlative super-resolution microscopy

doi: 10.1016/j.xpro.2024.103160

Figure Lengend Snippet: Image analysis pipeline (A) PALM acquisitions consist of 4–6 temporal streams of 2,000 images each, containing the fluorescence signals from both mEos3.2 fusion proteins and Tetraspeck beads. (B) Image stacks are then opened in SMLM software such as PALMTracer or ThunderSTORM. By setting a high detection threshold, one bead is chosen to correct for lateral drift. Then, by setting a lower detection threshold, single mEos3.2 molecules are detected, and a localization file is created, after applying the drift correction to all molecules. A single super-resolved image is created with a zoom factor of 5 (PALM image). (C) STED acquisitions give a single image from the Atto647N signal recognizing GFP-actin by immunocytochemistry (Ab-Atto647N). Because of potential depth in the sample, not all beads emit homogeneously. Thus, a small 3D stack is acquired in both TIRF and confocal modes (PALM and STED panels, respectively) and a maximal intensity projection is created to image all the beads equivalently. The SMLM software is used to create two separate localization files from the bead projection images obtained in PALM and STED, respectively. (D) Those files are then opened in Napari-bead-reg to create an image transformation matrix, which is applied to the single mEos3.2 molecule localization file, creating a transformed localization file. A new PALM image can then be generated with the SMLM software and super-imposed to the STED image. (E) FIJI is used to segment dendritic spines from STED images and calculate their projected area. The ROI drawn for each spine is exported to SR-Tesseler. (F) The SR-Tesseler software is then used to highlight the nanodomains formed in PALM images by some proteins such as PSD-95, count their number per spine, and measure their size. (G) Finally, the data are computed for a few dual PALM-STED acquisitions on individual neurons, and statistics are made.

Article Snippet: The image stacks obtained in PALM were analyzed using the PALMTracer software, a MetaMorph add-on., , Single molecule localization was achieved using wavelet segmentation, then filtered out based on the quality of a 2D Gaussian fit.

Techniques: Fluorescence, Software, Immunocytochemistry, Transformation Assay, Generated

Journal: STAR Protocols

Article Title: Protocol for matching protein localization to synapse morphology in primary rat neurons by correlative super-resolution microscopy

doi: 10.1016/j.xpro.2024.103160

Figure Lengend Snippet:

Article Snippet: The image stacks obtained in PALM were analyzed using the PALMTracer software, a MetaMorph add-on., , Single molecule localization was achieved using wavelet segmentation, then filtered out based on the quality of a 2D Gaussian fit.

Techniques: Recombinant, Modification, Saline, Staining, Plasmid Preparation, Software, Filtration, Electron Microscopy, Imaging, Microscopy