August 2011

The Roysam lab focused on pipeline integration in order to reveal kinks in the planned workflow.

Image Preprocessing Additions

• A background subtraction step to remove the low-frequency noise and large imaging artifacts from the raw images.
• A normalization step to equalize foreground contrast and correct for changes in overall brightness as a function of depth.

These additions allowed us to montage a few data sets which had previously defied automated registration.

Tiling Functions for Microglia Tracing and Nuclei Segmentation:

• Montage can be split into smaller tiles for processing.
• Workflow of individual tiles can be distributed more easily:
  • Tracing microglial tiles
  • Segmentation and classification of nuclei tiles
• Resulting files can be stitched back into global coordinates automatically, since the output of each tile only needs a static translation.

Soma Detection and Segmentation Improvements

• Bug: previous method of morphological functions on the microglia channel missed some somata.
• Fix: combining the DAPI channel with the microglia channel reduced extrasomatic noise, allowing us to use more relaxed parameters to identify somata.