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Functional fluorophores represent an emerging research field, distinguished by their diverse applications, especially in sensing and cellular imaging. After the discovery of quinine sulfate and subsequent elucidation of the fluorescence mechanism by Sir George Stokes, research in the field of fluorescence gained momentum. Over the past few decades, advancements in sophisticated instruments, including super-resolution microscopy, have further promoted cellular imaging using traditional fluorophores. These advancements include deciphering sensing mechanisms via photochemical reactions and scrutinizing the applications of fluorescent probes that specifically target organelles. This approach elucidates molecular interactions with biomolecules. Despite the abundance of literature illustrating different classes of probe development, a concise summary of newly developed fluorophores remains inadequate. In this review, we systematically summarize the chronological discovery of traditional fluorophores along with new fluorophores. We briefly discuss traditional fluorophores ranging from visible to near-infrared (NIR) in the context of cellular imaging and in vivo imaging. Furthermore, we explore ten new core fluorophores developed between 2007 and 2022, which exhibit advanced optical properties, providing new insights into bioimaging. We illustrate the utilization of new fluorophores in cellular imaging of biomolecules, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and proteins and microenvironments, especially pH and viscosity. Few of the fluorescent probes provided new insights into disease progression. Furthermore, we speculate on the potential prospects and significant challenges of existing fluorophores and their potential biomedical research applications. By addressing these aspects, we intend to illuminate the compelling advancements in fluorescent probe development and their potential influence across various fields.

A unique and highly water-soluble ICT-based fluorescent probe is developed for efficient detection and discrimination of reactive monocarbonyl formaldehyde (FA) from dicarbonyl methylglyoxal (MGO)/glyoxal (GO) by modulating the ICT process, which was confirmed by photophysical and TD-DFT analysis. The probe is applied in cellular imaging and quantifying FA in preserved food and MGO in manuka honey.

A 241-membered cyanine-based library was constructed by the combinatorial chemistry strategy. Combined with high-throughput screening, we successfully discovered a novel fluorescent probe (CyC-M716) capable of identifying a subset of parallel G-quadruplexes with propeller loops stretching across three tetrad layers with high sensitivity and selectivity.

A novel zwitterionic near-infrared (NIR) dye, ZWCC, has been designed and synthesized. It shows significantly enhanced photostability and chemical stability compared to the existing zwitterionic NIR dye. In addition, the feasibility of labeling ZWCC with biological ligands was investigated and used in live cell imaging applications.

The first fluorescent sensor for milk fat was developed. It exhibited a magnificent, yet selective turn-on feature towards fat molecules in a complicated milk matrix by a disaggregation-induced emission mechanism. Further construction of a handy fluorescence milk fat detector provided a convenient rapid tool to measure the fat amount quantitatively. This discovery may help enhance the milk quality control process.

Visualization of macrophages in live animals has been of great interest for a better understanding of inflammation. We developed a near infrared (NIR) probe that can selectively detect macrophages and visualize inflammation in vivo using the IVIS spectrum, Fluorescence Molecular Tomography (FMT) and Multi-Spectral Optoacoustic Tomography (MSOT).

SERS nanotags have been prepared to accomplish the multiplex detection of cancer cells. Herein we evaluated the adequacy of lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA) to function as multiplex partners with a triphenylmethine Raman reporter (B2LA) under a single excitation wavelength. SERS experiments enabled the multiplex recognition of two different cancer cells with antibody-conjugated nanotags that were derivatized with optimized cyanine and triphenylmethine reporters.

With the emerging interest in optical in vivo imaging, there is an increasing demand of photostable near-infrared (NIR) dyes. Herein we report the rational design of an amine tricarbocyanine structure with improved photostability (CyNA) and its combinatorial derivatization to render CyNA-414 as a NIR-fluorescent dye with stronger emission intensity and higher photostability than the NIR standard IndoCyanine Green (ICG).