Introduction: Fluorescent Probes in Biology

Published as part of Chemical Reviews special issue “Fluorescent Probes in Biology”. Seeing is believing! Chemistry makes the invisible visible through the use of imaging agents that can monitor the elements and molecules of life at a variety of length scales with both spatial and temporal resolution. In this context, fluorescence and luminescence provide visual readouts that can be widely adopted by specialists and nonspecialists alike, from laboratory researchers to medical clinicians and field technicians to children playing with glowsticks and fireflies during the long nights of summer. This thematic issue, Fluorescent Probes in Biology, delves into the latest research achievements in this storied and highly active field in the design and development of chemical reagents to decipher new fundamental biology and to translate this knowledge to advanced diagnostic and/or therapeutic platforms. Each paper in this issue focuses on molecular principles of probe design, applied to a particular biological question for analyte detection, chemical platform consisting of a small molecule, macromolecule, nanomaterial, or hybrid scaffold, or disease biomarker. State-of-the art research and future prospects in unmet needs pervade all these informative reviews. A key emerging theme in this collection is the broad use of activity-based sensing, a termed coined by our laboratory and advanced by our team and many others across the world, which is defined as using molecular reactivity rather than molecular recognition for analyte detection. (1) We organize this discussion across fluorescent probes for specific bioanalytes, fluorescent probes derived from a specific type of scaffold (e.g., small molecule, protein, nanomaterial, or hybrid), or fluorescent probes for biomedical applications. A foundational use of fluorescent probes in biology is their application in detecting the chemistry of elements and molecules of life to decipher their physiological and/or pathological contributions to living systems. New and our laboratory have collaborated to write a review on small-molecule sensors for transition metal ions in biological specimens (10.1021/acs.chemrev.3c00819). A focus is on the use of binding-based sensing and activity-based sensing approaches, where the former strategy exploits traditional lock-and-key metal–ligand coordination bonding for selective metal chelation and detection, whereas the latter concept leverages the diverse reactivity of metal ions for their sensing. These approaches are applied to image bioavailable metal pools, termed the labile metal pool, over a variety of biological length scales and in a variety of cell and animal models with metal and oxidation state selectivity, revealing new biological concepts such as transition metal signaling and metalloallostery in health and disease. Activity-based sensing offers a powerful approach to use reaction chemistry as a strategy for analyte detection. Lippert and Domaille and their teams review activity-based sensing strategies for reactive oxygen species (ROS) and reactive nitrogen species (RNS) across a diverse array of reaction scaffolds. These families of transient small molecules are toxic and lead to oxidative stress and oxidative damage when overproduced in aberrant quantities, but privileged members such as nitric oxide and hydrogen peroxide are also potent signaling agents that modify proteins, nucleic acids, and other biological targets with high specificity when produced in the right time and space. A key design feature is to develop reactions with selective one- and two-electron chemistry, as many of these ROS and RNS operate by free radical chemistry. Pluth and his colleagues write an excellent piece on activity-based sensing approaches on hydrogen sulfide and related reactive sulfur species (10.1021/acs.chemrev.3c00683). Hydrogen sulfide (H₂S) is an important representative of the ever-growing class of gasotransmitters along with nitric oxide, ammonia, carbon monoxide, carbon dioxide, and ethylene, as well as the primary reactive sulfur species. This review scholarly outlines reaction types for use in selective reactive sulfur species detection, from reduction of oxidized nitrogen motifs, electrophilic reactions, metal precipitation and metal coordination, and disulfide exchange. These tools have revealed a central role for H₂S and reductant-labile and sulfane sulfur species, including persulfides and polysulfides, as signaling agents that act by protein post-translational modifications. Zhang and Liu showcase design and application of fluorescent probes for physical microenvironments within cellular contexts (10.1021/acs.chemrev.3c00573). Foundational parameters such as pH, temperature, voltage, mechanical force, polarity, and viscosity offer physical markers that pervade all cells across all kingdoms of life. Fluorescent probes can be used to visualize dynamic changes in the cellular environment and be leveraged to expand fundamental knowledge and enrich diagnostic tools to better human health. Fluorescent probes can be constructed from a variety of chemical components that span synthetic small molecules to biological macromolecules to materials. Moreover, combinations of these basic building blocks can give rise to unique sensor platforms. Beyond traditional small-molecule organic fluorophores, Lo and colleagues review transition metal complexes that possess requisite optical properties and biocompatibility for bioimaging. These imaging agents often exhibit high photostability derived from complexes with inert metal–ligand bonds and can be used for activity-based sensing applications for bioanalyte detection along with protein and organelle imaging. Demirer and Beyene and their laboratories provide a comprehensive overview of fluorescent carbon nanomaterials and their applications for biomedical and environmental imaging, sensing, and cargo delivery applications (https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00581). Graphene and graphene oxide, carbon nanotubes and related carbon nanohoops, along with carbon dots showcase the range of materials that exhibit interesting photophysics for such applications. Zhang and his team write on nanocrystals for bioimaging (10.1021/acs.chemrev.3c00506), focusing on nanocrystal designs that absorb and emit in the near-infrared region (700–1700 nm). This optical profile enables deep tissue penetration that is necessary for in vivo imaging applications. Nanocrystals can be tailored for fluorescence, bioluminescence, and chemiluminescence imaging in intensity and time-resolved modes. Moreover, advanced sensor design by controlling energy transfer pathways gives rise to Cerenkov luminescent, X-ray excited luminescent, and persistent luminescent imaging. Kikuchi and colleagues review the exciting field of hybrid small-molecule/protein fluorescent probes (https://pubs.acs.org/doi/10.1021/acs.chemrev.3c00549), where these systems combine the molecular tunability of small-molecule reagents via chemical synthesis with biological scaffolds that enable site-specific delivery and augmented compatibility with living systems. This work is built upon the myriad of site-selective protein labeling reactions using exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemistry. Indeed, imaging of protein trafficking and conformational changes and bioanalytes with high signal-to-noise responses can be achieved using these hybrid platforms. In addition to fluorescent probes as reagents for learning about fundamental biology, they can also be applied in advanced diagnostic applications for medicine. Urano and Fujita write on activity-based fluorescence detection in cancer (10.1021/acs.chemrev.3c00612). These diverse classes of probes take advantage of biomarkers that are intrinsically elevated in various cancers. By administering an activatable probe to a disease specimen, from whole blood and serum to tumor tissue, a fluorogenic response can enable rapid cancer imaging with high contrast. A distinguished international team of Gunnlaugsson, James, Tang, Li, and Lewis and their colleagues provides a broad overview of fluorescent probes for disease diagnostics (10.1021/acs.chemrev.3c00776). They emphasize the design strategies behind these reagents, which open the door to applications in detecting bioactive molecules associated with diseases spanning organ damage, inflammation, cancers, cardiovascular diseases, and brain disorders. They also highlight future unmet needs to achieve accurate detection and identification of biomarkers for biomedical research with activity-based sensing reagents. Another large international collaboration between Kim, Sessler, Peng, James, Zeng, He, and Sharma and their laboratories focuses on theranostic fluorescent probes (10.1021/acs.chemrev.3c00778). This large field leverages the ability of activity-based sensing triggers that are activated by disease biomarkers to achieve spatiotemporal control in the context of drug delivery. When combined with a concomitant imaging response, these dual theranostic agents can achieve both targeted therapeutic delivery of a medicine and imaging readout. Indeed, photodynamic, photothermal, and sonodynamic therapeutic applications are but some of the numerous possibilities afforded by this theranostic approach using small-molecule to nanomaterial scaffolds. I hope that this collection of reviews will be a useful resource to introduce the exciting world of fluorescent probes to a broad range of newcomers as well as experts in the field, where chemistry can light the way to new knowledge and societal benefit. C.J.C. thanks the NIH (GM 79465, GM 139245, ES 28096) for support. Christopher J. Chang is the Edward and Virginia Taylor Professor of Bioorganic Chemistry at Princeton University. He completed his B.S. and M.S. degrees from Caltech in 1997 with Harry Gray, a Fulbright scholarship with Jean-Pierre Sauvage, a Ph.D. from MIT in 2002 with Dan Nocera, and a postdoc at MIT with Steve Lippard. Chris started his independent career at UC Berkeley in 2004 before moving to Princeton in 2024. The Chang laboratory focuses on metals in biology and energy. His group has pioneered the concept of activity-based sensing, showing that selectivity in sensor design is achievable by reaction-based methods that go beyond traditional receptors that operate by lock-and-key binding. His laboratory’s work has changed dogma in the inorganic and chemical biology communities by showing that transition metals are not merely active site cofactors in proteins but also serve as dynamic, allosteric regulators of protein function through metalloallostery, launching a field of transition metal signaling. This article references 1 other publications. This article has not yet been cited by other publications.