The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).

Fluorescence imaging has emerged as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution. Researchers have constructed a large number of synthetic intensity-based fluorescent probes for bio-imaging. However, intensity-based fluorescent probes have some limitations: variations in probe concentration, probe environment, and excitation intensity may influence the fluorescence intensity measurements. In principle, the use of ratiometric fluorescent probes can alleviate this shortcoming. Förster resonance energy transfer (FRET) is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. However, the development of synthetic FRET probes with favorable photophysical properties that are also suitable for biological imaging applications remains challenging. In this Account, we review the rational design and biological applications of synthetic FRET probes, focusing primarily on studies from our laboratory. To construct useful FRET probes, it is a pre-requisite to develop a FRET platform with favorable photophysical properties. The design criteria of a FRET platform include (1) well-resolved absorption spectra of the donor and acceptor, (2) well-separated emission spectra of the donor and acceptor, (3) donors and acceptors with comparable brightness, (4) rigid linkers, and (5) near-perfect efficiency in energy transfer. With an efficient FRET platform in hand, it is then necessary to modulate the donor-acceptor distance or spectral overlap integral in an analyte-dependent fashion for development of FRET probes. Herein, we emphasize our most recent progress on the development of FRET probes by spectral overlap integral, in particular by changing the molar absorption coefficient of the donor dyes such as rhodamine dyes, which undergo unique changes in the absorption profiles during the ring-opening and -closing processes. Although partial success has been obtained in design of first-generation rhodamine-based FRET probes via modulation of acceptor molar absorption coefficient, further improvements in terms of versatility, sensitivity, and synthetic accessibility are required. To address these issues with the first-generation rhodamine-based FRET probes, we have proposed a strategy for the design of second-generation probes. As a demonstration, we have developed FRET imaging probes for diverse targets including Cu²⁺, NO, HOCl, cysteine, and H₂O₂. This discussion of the methods for successfully designing synthetic FRET probes underscores the rational basis for further development of new FRET probes as a molecular toolbox for probing and manipulating a wide variety of biomolecules in living systems.

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H(2)O(2) and NIR-thiol, for H(2)O(2) and thiols, respectively, were constructed. These novel sensors respond to H(2)O(2) or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H(2)O(2) and NIR-thiol are capable of imaging endogenously produced H(2)O(2) and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.

Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.