Figure 5. Conceptual diagram of the dynamic changes in biomolecules during long-term storage in fixative.

During the initial fixation process, three primary states are identified: biomolecules covalently bonded into the biomolecule-aldehyde crosslinking mesh (termed “crosslinked”, black); biomolecules confined by the steric properties of this mesh without being covalently bonded (termed “confined”, grey); and aldehyde molecules, which can be either part of intramolecular or intermolecular crosslinks, bonded to a biomolecule as an adduct without forming a crosslink, or free in solution (purple). Here, the term “biomolecules” primarily refers to proteins, lipids, nucleic acids, and other macromolecules. Bioelements, although not biomolecules per se, are also included in this category. Regarding long-term storage alterations, there is: a general increase in the number of crosslinks; a conversion of many confined biomolecules to crosslinked states; hydrolysis of some existing crosslinks (red); fragmentation of certain biomolecules (blue); migration or leaching, which primarily happens to confined biomolecules (yellow); and chemical alterations of biomolecules, which could be due to composition or conformation changes (green). Note that the relative proportion of each alteration is not meant to be precise but solely for illustration purposes.