In vivo luminescence imaging employs the contrast generated by photoluminescent agents (known as luminophores) to visualize biological processes in living mammals. This technique proves invaluable for uncovering intricate physiological and pathological mechanisms within the native environment, owing to its high sensitivity, high resolution, real-time, and multiplexed capabilities [1,2]. The central challenge in this field lies in acquiring deep-tissue biological information labeled by luminophores. Since mammalian tissues are heterogeneous and inherently opaque to light, this fuels a demand to identify an optimal spectral window for developing luminophores that enhance light penetration and contrast [3, 4, 5].

By the 1980s, the biological community acknowledged the benefits of near-infrared (NIR) light in tissue examinations [6]. The NIR-II window, in the wavelength range of 1000–2000 nm emerged after 2009 under the explorative use of InGaAs or TeCdHg array detectors and superconducting nanowire single-photon detectors [7, 8, 9, 10, 11]. This surpassed the conventional visible (400–700 nm) and NIR-I (700–1000 nm) window, offering reduced tissue scattering (Figure 1a) and autofluorescence (Figure 1b) at longer wavelengths. However, within the NIR-II window, the penetration depth is wavelength-dependent due to water overtone absorption (Figure 1a), led to the precise definition of distinct sub-windows over the past decade (Figure 1c). While attenuating signal intensity, water absorption enhances contrast by suppressing scattered photons [5]. As such, we have simplified the nomenclature of NIR-II sub-windows to the NIR-II short-wavelength (NIR–II–S; 1000–1400 nm) and NIR-II long-wavelength (NIR–II–L; 1500–1900 nm) windows, based on their actual imaging performance. NIR–II–S with minimized water absorption, is suitable for large-scale structure imaging, such as organ-level imaging, facilitating the capture of deep tissue signals. On the other hand, NIR–II–L with moderate water absorption is preferable for high-contrast imaging of small-scale structures like cells and vessels.

The exploration of the NIR-II window has attracted considerable interest in developing novel luminophore systems (Figure 1c). The first imaging demonstration employed single-walled carbon nanotubes (SWCNTs), which revealed the varied benefits of the NIR-II sub-windows across the 1000–1700 nm range by harnessing their broad spectral tunability [3,4,8]. Since then, quantum dots [10,12] and lanthanide-doped nanocrystals [13, 14, 15, 16] have been successively developed and explored. These inorganic nanomaterials provide significant advantages, including extremely high luminescence quantum yields, narrow bandwidths, and wavelength tunability across an extended range of 1000–2000 nm. These features facilitate the tracking of rapid dynamic biological processes and capitalize on the broad NIR-II spectrum for multiplexed imaging. However, the functionalization and bioconjugation of these materials often require complex procedures, and concerns about their potential physiological toxicity persists. In contrast, small-molecule dyes, with their long research history, have been fully proven effective and biocompatible for labeling and imaging biomolecules, cells, or tissues, thereby supporting their potential for clinical translation. For example, Indocyanine Green (ICG) has successfully been used as a contrast agent in clinical applications, and several functionalized IRDye800CW agents are currently undergoing clinical trials [17, 18, 19]. In 2015, the Dai group introduced CH1055, the first NIR-II small molecule dye, inspiring significant efforts to develop new molecular structures, driven by a persistent ambition to achieve longer wavelengths and higher brightness [20, 21, 22, 23]. Yet, there's a current lack of small-molecule dyes that achieve peak emission in the NIR–II–L window, posing challenges for the comprehensive exploration of the entire NIR-II window using molecular science and engineering.

In this context, lanthanide-dye hybrid luminophores (LDHLs) featuring a basic molecular structure comprising a dye ligand and a trivalent lanthanide ion (Figure 2a), represent a novel luminophore system that integrates the traits of small molecule dyes and lanthanide-doped nanocrystals, offering molecular size and distinct spectra with sharp fingerprint emission in the NIR–II–S and NIR–II–L windows. These characteristics create substantial possibilities for exploration in in vivo dynamic multiplexed imaging and clinical translation. This minireview aims to provide a concise introduction to LDHLs and their applications in in vivo imaging and analysis. Additionally, we explore the challenges linked to the rational design of novel LDHL materials, with the aspiration to inspire the community to develop and apply them in new areas.