Smart fluorescent probes for macrophage activity make use of a broad range of chemical architectures to target different molecular targets.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small‐sized multi‐colored fluorophores for real‐time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.

Immunomodulatory agents represent one of the most promising strategies for enhancing tissue regeneration without the side effects of traditional drug-based therapies. Tissue repair depends largely on macrophages, making them ideal targets for proregenerative therapies. However, given the multiple roles of macrophages in tissue homeostasis, small molecule drugs must be only active in very specific subpopulations. In this work, we have developed the first prodrug–fluorophore conjugates able to discriminate closely related subpopulations of macrophages (i.e., proinflammatory M1 vs anti-inflammatory M2 macrophages), and employed them to deplete M1 macrophages in vivo without affecting other cell populations. Selective intracellular activation and drug release enabled simultaneous fluorescence cell tracking and ablation of M1 macrophages in vivo, with the concomitant rescue of a proregenerative phenotype. Ex vivo assays in human monocyte-derived macrophages validate the translational potential of this novel platform to develop chemical immunomodulatory agents as targeted therapies for immune-related diseases.

Herein tricarbocyanine N-triazoles are first described as a rationally-designed near-infrared (NIR) structure overcoming the brightness and photostability limitations of tricarbocyanines for long-term in vivo imaging. The straightforward synthetic approach and the wide availability of alkynes makes this strategy a versatile methodology for the preparation of highly stable N-substituted tricarbocyanines. Furthermore, we validated CIR38M as a non-transferable marker to monitor the fate of therapeutic T cells non-invasively in vivo, showing enhanced performance over conventional NIR fluorophores (i.e.DiR, IR800CW and indocyanine green) as well as compatibility with human cells for translational studies. CIR38M is able to track over time smaller numbers of T cells than current NIR agents, and to visualise antigen-driven accumulation of immune cells at specific sites in vivo. This chemical technology will improve longitudinal imaging studies to assess the efficacy of cell-based immunotherapies in preclinical models and in human samples.

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND‐gates since they emit only after receptor binding and intracellular activation, showing enhanced selectivity over existing agents. We have applied this strategy to produce mCCL2‐MAF as the first probe for in vivo detection of metastasis‐associated macrophages in a preclinical model of lung metastasis. This strategy will accelerate the preparation of new chemokine‐based probes for imaging immune cell function in tumours.