Quantification of Dendritic Spines Using Confocal Microscopy Imaging

Take pyramidal neurons that are transduced and immunolabeled to fluoresce green.

Acquire images of the neurons using a confocal laser-scanning microscope with settings adjusted to ensure optimal image quality.

Open an image using specialized software and use the Gaussian filter to reduce the background noise.

Perform semi-automatic dendrite tracing by first estimating the dendrite diameter. Then, select the dendrite's starting point, path, and endpoint.

For automated spine segmentation, select rebuild dendrite diameter and set dendrite volume threshold to represent the actual dendrite volume.

Specify values for the smallest spine head diameter and the maximal spine length.

Adjust the threshold for points representing spines, ensuring their localization on actual spine heads.

Set parameters to classify the spines based on length, head diameter, and shape.

Generate a 3D model of the dendrite with spines and export the data for further analysis.

Under a confocal microscope, select at least 10 healthy neurons per condition with a pyramidal morphology and a full dendritic arborization. To quantify 60 to 100 micrometers per dendrite, acquire images with a 40x, NA 1.3 oil objective and a 488-nano