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. 2018 Mar 29;12:80. doi: 10.3389/fncel.2018.00080

Figure 1.

Figure 1

Spatially resolved NAnoparticle-enhanced Photoporation (SNAP) enables fast and targeted labeling of primary hippocampal neurons and their spines. (A) General workflow of SNAP. Intracellular delivery of phalloidin relies on the adsorption of cell-interactive plasmonic gold nanoparticles (AuNP) to the plasma membrane. Upon selective illumination of the cell of interest with 561 nm laser light, AuNPs heat up and induce vapor nanobubbles (VNBs). These VNBs transiently open the membrane, allowing the otherwise impermeable dye to enter the soma (white arrows). After washing and allowing time for intracellular diffusion of the dye to the neuronal extremities, completely labeled neurons can be monitored with high contrast. (B) Workflow of image-guided SNAP. Before the actual SNAP procedure is initiated, large field-of-view images are acquired of cells labeled with a cell-permeable marker. After image analysis and selection, the coordinates of the cells of interest are determined and fed into the SNAP setup for targeted photoporation. (C) Image-guided SNAP on primary neurons. Nuclei are stained with the membrane-permeable dye Hoechst. Using image analysis, all nuclei are first detected, followed by a selection of nuclei that are at least 200 μm apart (gold circles and arrowheads). The coordinates of the neurons of interest are used to guide the SNAP procedure. The high-resolution image of the phalloidin-labeled neuron shows dendritic spines (arrows) in high contrast without interference of fluorescence from overlapping dendrites or axons of nearby cells.