Investigation of the physiological and pathological functions of formaldehyde (FA) are largely restricted by a lack of useful FA imaging agents, in particular, those that allow detection of FA in the context of living tissues. Herein, we present the rational design, synthesis, and photophysical property studies of the first two-photon fluorescent FA probe, Na-FA. Importantly, the highly desirable attributes of the probe Na-FA (such as a very large turn-on signal (up to 900-fold), a low detection limit, and a very fast onset imparted by the unique design aspects of the probe), make it possible to monitor endogenous FA in living tissues for the first time. Furthermore, sodium bisulfite was identified as a simple and convenient inhibitor of FA within biological environments.

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.

Fluorescence imaging is a powerful approach for noninvasive and real-time visualization and tracking of biomolecules and biological processes in living systems. The fluorescent chemosensors with dual/triple interplaying sensing mechanisms tend to provide diverse fluorescence signals or amplify the response signals, which are propitious to simultaneously track multiple analytes or to improve the selectivity and sensitivity of the chemosensors. Thus, the development of dual/triple sensing mechanism-based chemosensors has attracted great interest recently. This review highlights the representative cases of the fluorescent chemosensors with dual/triple interplaying sensing mechanisms published since 2010, and these chemosensors are classified according to the types of the interplaying sensing mechanisms, including ICT-FRET, PET-FRET, PET-ICT-ESIPT, etc.

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.

A new ratiometric fluorescent H O probe, benzopyrylium-coumarin (BC), is designed by using an oxonium moiety as the unique H O response site. The BC probe exhibits an extremely large emission shift of 221 nm in response to H O , and is successfully applied for the simultaneous near-infrared and two-photon imaging of H O in living cells, mouse-liver tissues, and zebrafish.