The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer's disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes-regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.