S1. DISCO-MS pipeline
Step-by-step DISCO-MS pipeline illustrated on a 5xFAD mouse brain (6 weeks old). LCM extracted ROIs around the single plaques subjected to ultra-high sensitive mass spectrometry based proteome analyses.
S2. DISCO-bot illustration
Robotic arm illustration showing the steps of extraction from DISCO/SHANEL cleared whole mouse and large human organs. DISCO-bot uses the biopsy needles of various sizes to extract the ROIs for the subsequent molecular analysis.
S3. Mild traumatic brain injury model in CX3CR1-EGFP brains
vDISCO labeled and cleared CX3CR1-EGFP brain showing microglia activation in a region-specific manner after mTBI compared to sham. Particularly, highlighted regions are along the corpus callosum and optic tract (segmented microglia in magenta).
S4. Segmented plaques in Alzheimer’s disease model
5xFAD whole mouse brain showing deep learning-based segmentation of plaques at later stage of disease (6 months old).
S5. DISCO-bot real-time extraction of ROIs from scapula of mouse
Robotic arm guided real time extraction of ROIs from bone marrow of scapula in mouse. The procedure involves 4 major steps: 1) Finding the target of interest and approaching with needle 2) cutting through bone to reach the marrow 3) excising around the target and extracting the tissue with vacuum, and 4) examining the extracted area to ensure successful extraction.
S6. DISCO-bot extracted ROIs from whole body of a cleared LysM-EGFP mouse
DISCO-bot ROIs extraction from the scapula and cranium of a LysM-EGFP mouse cleared with vDISCO. LysM immune cells are shown in magenta; PI labelled nuclei are in yellow and autofluorescence is in cyan.
S7. Spatial identification of atherosclerotic plaques along the coronary artery
Atherosclerotic human heart after SHANEL clearing and imaging, showing the plaque vs. non-plaque regions along the right coronary artery. Plaques are segmented in gold; vessels are in magenta and autofluorescence is in cyan.