Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for physiological studies and biomedical applications. However, inhomogeneous signal attenuation in biological matter hampers the application of multiple-wavelength NIR-II probes to multiplexed imaging. Here, we present lanthanide-doped NIR-II nanoparticles with engineered luminescence lifetimes for in vivo quantitative imaging using time-domain multiplexing. To achieve this, we have devised a systematic approach based on controlled energy relay that creates a tunable lifetime range spanning three orders of magnitude with a single emission band. We consistently resolve selected lifetimes from the NIR-II nanoparticle probes at depths of up to 8 mm in biological tissues, where the signal-to-noise ratio derived from intensity measurements drops below 1.5. We demonstrate that robust lifetime coding is independent of tissue penetration depth, and we apply in vivo multiplexing to identify tumour subtypes in living mice. Our results correlate well with standard ex vivo immunohistochemistry assays, suggesting that luminescence lifetime imaging could be used as a minimally invasive approach for disease diagnosis.

The contrast and sensitivity of in vivo fluorescence imaging has been revolutionized by molecular fluorophores operating in the second near-infrared window (NIR-II; 1000-1700 nm), but an ongoing challenge is the solvatochromism-caused quenching in aqueous solution for the long-wavelength absorbing fluorophores. Herein, we develop a series of anti-quenching pentamethine cyanine fluorophores that significantly overcome the severe solvatochromism, thus affording stable absorption/emission beyond 1000 nm with up to ~ 44-fold enhanced brightness and superior photostability in aqueous solution. These advantages allow for deep optical penetration (8 mm) as well as high-contrast and highly-stable lymphatic imaging superior to clinical-approved indocyanine green. Additionally, these fluorophores exhibit pH-responsive fluorescence, allowing for noninvasive ratiometric fluorescence imaging and quantification of gastric pH in vivo. The results demonstrate reliable accuracy in tissue as deep as 4 mm, comparable to standard pH electrode method. This work unlocks the potential of anti-quenching pentamethine cyanines for NIR-II biological applications.

Local recurrence is a common cause of treatment failure for patients with solid tumors. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and, thereby improve prognosis. Here, we report in vivo assembly of the second near-infrared window (NIR-II) emitting downconversion nanoparticles (DCNPs) modified with DNA and targeting peptides to improve the image-guided surgery for metastatic ovarian cancer. The NIR-II imaging quality with DCNPs is superior to that of clinically approved ICG with good photostability and deep tissue penetration (8 mm). Stable tumor retention period experienced 6 h by in vivo assembly of nanoprobes can be used for precise tumor resection. Superior tumor-to-normal tissue ratio is successfully achieved to facilitate the abdominal ovarian metastases surgical delineation. Metastases with ≤1 mm can be completely excised under NIR-II bioimaging guidance. This novel technology provides a general new basis for the future design of nanomaterials for medical applications.

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Fluorescent bioimaging in the second near-infrared window (NIR-II) can probe deep tissue with minimum auto-fluorescence and tissue scattering. However, current NIR-II fluorophore-related biodetection in vivo is only focused on direct disease lesion or organ bioimaging, it is still a challenge to realize NIR-II real-time dynamic biosensing. A new type of Er sensitized upconversion nanoparticles are presented with both excitation (1530 nm) and emission (1180 nm) located in the NIR-II window for in vivo biosensing. The microneedle patch sensor for in vivo inflammation dynamic detection is developed based on the ratiometric fluorescence by combining the effective NIR-II upconversion emission and H O sensing organic probes under the Fenton catalysis of Fe . Owing to the large anti-Stokes shifting, low auto-fluorescence, and tissue scattering of the NIR-II upconversion luminescence, inflammation can be dynamically evaluated in vivo at very high resolution (200×200 μm).

Deep tissue imaging in the second near‐infrared (NIR‐II) window holds great promise for widespread fundamental research. However, inhomogeneous signal attenuation due to tissue absorption and scattering hampers its application for accurate in vivo biosensing. Here, lifetime‐based in situ hepatocellular carcinoma (HCC) detection in NIR‐II region is presented using a tumor‐microenvironment (peroxynitrite, ONOO−)‐responsive lanthanide–cyanine Förster resonance energy transfer (FRET) nanosensor. A specially designed ONOO−‐responsive NIR‐II dye, MY‐1057, is synthesized as the FRET acceptor. Robust lifetime sensing is demonstrated to be independent of tissue penetration depth. Tumor lesions are accurately distinguished from normal tissue due to the recovery lifetime. Magnetic resonance imaging and liver dissection results illustrate the reliability of lifetime‐based detection in single and multiple HCC models. Moreover, the ONOO− amount can be calculated according to the standard curve.

Quantitatively imaging the spatiotemporal distribution of biological events in living organisms is essential to understand fundamental biological processes. Self-calibrating ratiometric fluorescent probes enable accurate and reliable imaging and sensing, but conventional probes using wavelength of 400–900 nm suffer from extremely low resolution for in vivo application due to the disastrous photon scattering and tissue autofluorescence background. Here, we develop a NIR-IIb (1500–1700 nm) emissive nanoprobe for high-resolution ratiometric fluorescence imaging in vivo. The obtained nanoprobe shows fast ratiometric response to hypochlorous acid (HOCl) with a detection limit down to 500 nM, through an absorption competition-induced emission (ACIE) bioimaging system between lanthanide-based downconversion nanoparticles and Cy7.5 fluorophores. Additionally, we demonstrate the superior spatial resolution of 1550 nm to a penetration depth of 3.5 mm in a scattering tissue phantom, which is 7.1-fold and 2.1-fold higher than that of 1064 and 1344 nm, respectively. With this nanoprobe, clear anatomical structures of lymphatic inflammation in ratiometric channel are observed with a precise resolution of ∼477 μm. This study will motivate the further research on the development of NIR-II probes for high-resolution biosensing in vivo.

In vivo fluorescence imaging in the second near‐infrared window (NIR‐II) affords deep‐tissue penetration and high spatial resolution. Herein, we present a new type of Tm3+‐sensitized lanthanide nanocrystals with both excitation (1208 nm) and emission (1525 nm) located in the NIR‐II window for in vivo optical information storage and decoding. Taking advantage of the tunable fluorescence lifetimes, the optical multiplexed encoding capacity is enhanced accordingly. Micro‐devices with QR codes featuring the NIR‐II fluorescence‐lifetime multiplexed encoding were implanted into mice and were successfully decoded through time‐gated fluorescence imaging technology.