Figure 2. Envisaged operational process of the proposed activatable ultracompact xanthene-core based molecular probe that demonstrates an OFF-ON NIR fluorescence response facilitated via NTR activity.

A low pO₂ environment corresponds to upregulated NTR activity. Step 1: The proposed NTR-selective activatable xanthene core-based molecular probe would diffuse across the cell membrane due to having a neutral net charge. Step 2: The hypoxia-induced (i) upregulated NTR activity in combination with (ii) suppressed oxidation back-reaction of its futile redox cycle would activate and maintain, respectively, its OFF-ON NIR fluorescence response by discontinuing and precluding a d-PeT fluorescence-quenching process from occurring. Step 3: The local equilibrium gradient would result in the activated molecular probe readily diffusing into lysosomes, wherein it would become trapped upon its fluorogenic scaffold assuming a positive net charge due to its integrated THQ moiety becoming weakly-protonated when in lysosomes. On doing so, the activated molecular probe would be permanently retained within the lysosomes. Step 4: The local equilibrium gradient would shift towards additional activated molecular probe localizing to the lysosomes, wherein it too would become similarly protonated and subsequently retained. This strategy would drive the retention of increasing amounts of activated probe, and thereby would help afford enhanced contrast levels due to the signal amplification resulting from such extensive activated molecular probe accumulation within the lysosomes. Ar = aromatic group, NTR = nitroreductase enzyme. pO₂ = partial pressure of oxygen.