Thioflavin T (ThT) exhibits an enormous increase in fluorescence intensity (or lifetime) upon binding to beta-sheet rich protein aggregates. In this letter, we measured the fluorescence lifetimes of ThT in poly (ethylene oxide) (PEO) and poly (acrylic acid) (PAA) polymer films and nanofibers and observed that the excited-state motion of ThT is slowed down as the mechanical rigidity of medium increases. Using ThT as a rigidity sensor, we first demonstrate that the fluorescence lifetime imaging microscopy (FLIM) can easily distinguish the PEO and PAA nanofiber structures, which were not discernable by electron microscopy. (C) 2013 Elsevier B. V. All rights reserved.