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Convenient and rational design of small molecular weight, water-soluble, two-photon excited fluorescence (TPEF) probes remains a challenge for practical biological applications. In this study, we designed six novel water-soluble, near infrared TPEF probes (NL1-3, PL1-3), bearing N-methyl pyridinium moiety, which are specifically sensitive to local viscosity. Fluorescence emission bands arid two-photon absorption cross-sections of the title compounds showed a significant enhancement with increased solvent viscosity. The compound NL1, a high fluorescent probe that selectively targeted the mitochondria and was sensitive to mitochondrial environment changes, could be applied to differentiate between nystatin treated and normal cell mitochondrial environment. Furthermore, NL1 is membrane potential independent, which was rarely found in contemporary probes. In addition to two-photon confocal microscopy, NL1 also displayed successful mitochondrial staining under stimulated emission depletion (STED) nanoscopy in living cells. In a nutshell, the merits of near infra-red region (NIR) operation, high selectivity, and signal ratio of these probes facilitate the study of mitochondrial targeting, localization processes, and mitochondria microenvironment related diseases. (C) 2017 Elsevier Ltd. All rights reserved.