Table 5.

Summary of expected preservation effects on key components of the biomolecule-annotated connectome.

Structural feature	Unprotected cryo-preservation	Cryo-preservation with CPAs	Fixation and cryo-preservation	Fluid preservation	Polymer embedding
Cell membrane shape	•Expected to be lost in many areas throughout the brain due to ice damage	•Could be retained, but ultrastructural preservation is unproven in whole mammalian brains	•Found to preserve the connectome via electron microscopy in the pig brain •Not yet shown in human brains	•Largely expected to be intact, with certain artifacts found over time in some studies (108)	•Largely expected to be intact using epoxy methods •But currently unable to infiltrate whole brain with epoxy
Proteins	•Mostly present •May aggregate, move, or change conformation with ice damage	•Mostly present •May have conformation changes due to CPAs, but likely minimal	•Mostly present •Both location and composition expected to be unchanged •Conformation may be altered	•Mostly present •May be change to chemical composition •Conformation may be altered	•Mostly present •Harsh solvent treatment could affect conformation and composition
Nucleic acids	•Mostly present, but may be damaged due to ice formation	•Mostly present, not expected to have major changes	•Mostly present •Molecules are fixed, so unable to dissociate them for DNA sequencing	•Mostly present •May accumulate damage over time, unclear degree	•Likely mostly present •Uncertain effects of processing on DNA content
Lipids	•Mostly present, but may be damaged due to ice formation	•Mostly present, not expected to have major changes	•Mostly present, not expected to have major changes	•Mostly retained but inaccessible •Subset may be lost or chemically altered	•Subset retained due to osmium fixation •But many lipids are expected to be extracted