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Conspectus Fluorescence imaging is a rapidly growing technique for noninvasive imaging of biological molecules and processes with high spatial and temporal resolution. For effective biological imaging, it is essential and important to develop robust fluorescent dyes, in particular, near-infrared (NIR) fluorescent dyes with favorable optical properties. Compared with the visible light emitting dyes, NIR dyes have relatively longer emission wavelengths (650-900 nm) with lower energy and are advantageous as imaging agents owing to the minimum photodamage of NIR light to biological samples, deep penetration into tissues, and low interference from autofluorescence of biomolecules. Although great efforts have been devoted to engineer NIR fluorophores, it is still very challenging to regulate their photophysical properties as they often lack optically tunable mechanisms, and this shortcoming considerably restricts the realization of their full potential. Consequently, the rational design of small-molecule optically tunable NIR fluorophores is of high priority and great value. In general, two key characteristics are indispensable for designing excellent optically tunable NIR fluorescent dyes. First, NIR fluorescent dyes should display the maximal absorption and emission located in the NIR region and also have the prominent properties including excellent fluorescence quantum yields, large Stokes shifts, good chemical stability and photostability, low cytotoxicity, and desirable compatibility with biological systems. Second, in principle, functional NIR dyes should also possess optically tunable groups, which can be easily modified to afford responsive sites for the targets of interest. With these considerations in mind, in this Account, we described a unique "integration" strategy for judicious design of the optically tunable NIR fluorophores, which are an intuitive combination of the traditional NIR dyes and the optically tunable mechanisms in the visible light emissive dyes. Thus, the versatile strategy may allow not only retention of the NIR emission properties of NIR dyes but also inheritance of the optically tunable mechanisms from the visible light emissive dyes. By the unique integration strategy, a built-in optically tunable group is strategically installed into the traditional NIR fluorescent dyes to directly tune their optical properties. Herein, we present a concise review of the rational design strategy and biological applications of small-molecule optically tunable NIR fluorescent dyes via the unique integration strategy, and we focused mainly on our work and some representative examples from other groups based on our NIR platforms. This Account includes the detailed integration strategy of each class of the NIR fluorescent dyes, the development of their derivatives, and their imaging applications in living systems. [ABSTRACT FROM AUTHOR]

Acidic pH is a critical physiological factor for controlling the activities and functions of lysosome. Herein, we report a novel dual site-controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-Fluorescence resonance energy transfer (ICT-PET-FRET) fluorescent probe (CN-pH), which was essentially the combination of a turn-on pH probe (CN-1) and a turn-off pH probe (CN-2) by a nonconjugated linker. Coumarin and naphthalimide fluorophores were selected as donor and acceptor to construct the FRET platform. Hydroxyl group and morpholine were simultaneously employed as the two pH sensing sites and controlled the fluorescence of coumarin and naphthalimide units by ICT and PET, respectively. The sensing mechanism of CN-pH to pH was essentially an integration of ICT, PET, and FRET processes. Meanwhile, the morpholine also can serve as a lysosome-targeted group. By combining the two data analysis approaches of the ratios of the two emission intensities (R) and the reverse ratio R-' (R' = 1/R), the fluorescent ratio of CN-pH can show proportional relationship to pH values in a very broad range from pH 4.0 to 8.0 with high sensitivity. The probe has been successfully applied for the fluorescence imaging of the lysosomal pH values, as well as ratiometrically visualizing chloroquine-stimulated changes of intracellular pH in living cells. These features demonstrate that the probe can afford practical application in biological systems. [ABSTRACT FROM AUTHOR]

Discrimination of live and dead cells is an important task in biological, pathological, medical, and pharmaceutical studies. In this work, we have developed a novel fluorescent probe DACA that can discriminate live and dead cells in a dual-color mode for the first time. DACA can stain dead cells with blue fluorescence peaked at 440 nm, while it can also label live cells with orange emission peaked at 570 nm. Compared with one-color fluorescent probes, such a dual-color probe can efficiently avoid false positive results from cellular autofluorescence and misleading signals brought by inhomogeneous staining, and thus can supply more accurate information in biological applications. By means of DACA, the health status of tumor cells pretreated by H2O2 and ultraviolet radiation has been successfully detected and imaged. Moreover, DACA and the hydrolyzed product exhibit excellent two-photon properties. Live and dead cells, as well as the zebrafishes, have been discriminated with dual emission colors under one- and two-photon microscope. These results demonstrate that DACA is a powerful tool for dual-color distinguishing live and dead cells in vitro and in vivo. [ABSTRACT FROM AUTHOR]