in vivo and in situ robust quantitative optical biopsy of hepatocellular carcinoma and metastasis based on two-photon autofluorescence imaging.

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.

Boosted photo-immunotherapy via near-infrared light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes.

Phototherapy is a promising modality that could eradicate tumor and trigger immune responses via immunogenic cell death (ICD) to enhance anti-tumor immunity. However, due to the lack of deep-tissue-excitable phototherapeutic agents and appropriate excitation strategies, the utility of phototherapy for efficient activation of the immune system is challenging. Herein, we report functionalized ICG nanoparticles (NPs) with the capture capability of tumor-associated antigens (TAAs). Under near-infrared (NIR) light excitation, the ICG NPs exhibited high-performance phototherapy, i.e., synergistic photothermal therapy and photodynamic therapy, thereby efficiently eradicating primary solid tumor and inducing ICD and subsequently releasing TAAs. The ICG NPs also captured TAAs and delivered them to sentinel lymph nodes, and then the sentinel lymph nodes were activated with NIR light to trigger efficient T-cell immune responses through activation of dendritic cells with the assistance of ICG NP generated reactive oxygen species, inhibiting residual primary tumor recurrence and controlling distant tumor growth. The strategy of NIR light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes provides a powerful platform for active immune systems for anti-tumor photo-immunotherapy.

Data-driven coordinated attention deep learning for high-fidelity brain imaging denoising and inpainting.

Deep learning offers promise in enhancing low-quality images by addressing weak fluorescence signals, especially in deep in vivo mouse brain imaging. However, current methods struggle with photon scarcity and noise within in vivo deep mouse brains, and often neglecting tissue preservation. In this study, we propose an innovative in vivo cortical fluorescence image restoration approach, combining signal enhancement, denoising, and inpainting. We curated a deep brain cortical image dataset and developed a novel deep brain coordinate attention restoration network (DeepCAR), integrating coordinate attention with optimized residual networks. Our method swiftly and accurately restores deep cortex images exceeding 800 µm, preserving small-scale tissue structures. It boosts the peak signal-to-noise ratio (PSNR) by 6.94 dB for weak signals and 11.22 dB for large noisy images. Crucially, we validate the effectiveness on external datasets with diverse noise distributions, structural features compared to those in our training data, showcasing real-time high-performance image restoration capabilities.

Biocompatible polymer optical fiber with a strongly scattering spherical end for interstitial photodynamic therapy.

Interstitial photodynamic therapy (I-PDT), which utilizes optical fibers to deliver light for photosensitizer excitation and the elimination of penetration depth limitation, is a promising modality in the treatment of deeply seated tumors or thick tumors. Currently, the excitation domain of the optical fiber is extremely limited, restricting PDT performance. Here, we designed and fabricated a biocompatible polymer optical fiber (POF) with a strongly scattering spherical end (SSSE) for I-PDT applications, achieving an increased excitation domain and consequently excellent in vitro and in vivo therapeutical outcomes. The POF, which was drawn using a simple thermal drawing method, was made of polylactic acid, ensuring its superior biocompatibility. The excitation domains of POFs with different ends, including flat, spherical, conical, and strongly scattering spherical ends, were analyzed and compared. The SSSE was achieved by introducing nanopores into a spherical end, and was further optimized to achieve a large excitation domain with an even intensity distribution. The optimized POF enabled outstanding therapeutic performance of I-PDT in in vitro cancer cell ablation and in vivo anticancer therapy. All of its notable optical features, including low transmission/bending loss, superior biocompatibility, and a large excitation domain with an even intensity distribution, endow the POF with great potential for clinical I-PDT applications.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

Superior biocompatible carbon dots for dynamic fluorescence imaging of nucleoli in living cells.

The nucleolus is a newly developed and promising target for cancer diagnosis and therapy, and its imaging is extremely significant for fundamental research and clinical applications. The unique feature, i.e., high resolution at the subcellular level, makes the fluorescence imaging method a powerful tool for nucleolus imaging. However, the fluorescence imaging of nucleoli in living cells is restricted by the limited availability of fluorescent agents with specific nucleolus-targeting capability and superior biocompatibility. Here, promising carbon dots (CDs) with intrinsic nucleolus-targeting capability were synthesized, characterized and employed for dynamic fluorescence imaging of nucleoli in living cells. The CDs exhibit a high fluorescence quantum yield of 0.2, excellent specificity and photostability, and superior biocompatibility, which were systematically demonstrated at the gene, cellular and animal levels and confirmed by their biological effects on embryonic development. All these features enabled CDs to light up the nucleoli for a long time with a high signal-to-noise ratio in living cells and monitor the nucleolar dynamics of malignant cells in camptothecin (CPT) based chemotherapy. Their excellent optical and biological features as well as general nucleolus-targeting capability endow CDs with great potential for future translational research.

The stiffness-dependent tumor cell internalization of liquid metal nanoparticles.

The properties of nanoparticle (NP) carriers, such as size, shape and surface state, have been proven to dramatically affect their uptake by tumor cells, thereby influencing and determining the effect of nanomedicine on tumor theranostics. However, the effect of the stiffness of NPs on their cellular internalization remains unclear, especially for circumstances involving active or passive NP targeting. In this work, we constructed eutectic gallium indium liquid metal NPs with the same particle size, shape and surface charge properties but distinct stiffness via tailoring the surface oxidation and silica coating. It has been found that the softer NPs would be endocytosed much slower than their stiffer counterparts in the presence of specific ligand-receptor interaction. Interestingly, once the interaction is eliminated, softer NPs are internalized faster than the stiffer ones. Based on experimental observations and theoretical verification, we demonstrate that this phenomenon is mainly caused by varying degrees of deformation of soft NPs induced by ligand-receptor interactions. Such a finding of the stiffness effect of NPs implies great potential for fundamental biomedical applications, such as the rational design of nanomedicines.

Light amplified oxidative stress in tumor microenvironment by carbonized hemin nanoparticles for boosting photodynamic anticancer therapy.

Photodynamic therapy (PDT), which utilizes light excite photosensitizers (PSs) to generate reactive oxygen species (ROS) and consequently ablate cancer cells or diseased tissue, has attracted a great deal of attention in the last decades due to its unique advantages. However, the advancement of PDT is restricted by the inherent characteristics of PS and tumor microenvironment (TME). It is urgent to explore high-performance PSs with TME regulation capability and subsequently improve the therapeutic outcomes. Herein, we reported a newly engineered PS of polymer encapsulated carbonized hemin nanoparticles (P-CHNPs) via a facile synthesis procedure for boosting photodynamic anticancer therapy. Solvothermal treatment of hemin enabled the synthesized P-CHNPs to enhance oxidative stress in TME, which could be further amplified under light irradiation. Excellent in vitro and in vivo PDT effects were achieved due to the improved ROS (hydroxyl radicals and singlet oxygen) generation efficiency, hypoxia relief, and glutathione depletion. Moreover, the superior in vitro and in vivo biocompatibility and boosted PDT effect make the P-CHNPs a potential therapeutic agent for future translational research.

Noninvasive and early diagnosis of acquired brain injury using fluorescence imaging in the NIR-II window.

Acquired brain injury (ABI), which is the umbrella term for all brain injuries, is one of the most dangerous diseases resulting in high morbidity and mortality, making it extremely significant to early diagnosis of ABI. Current methods, which are mainly composed of X-ray computed tomography and magnetic resonance angiography, remain limited in diagnosis of ABI with respect to limited spatial resolution and long scanning times. Here, we reported through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy, utilizing the fluorescence of down-conversion nanoparticles (DCNPs) in the 1.3 - 1.7 µm near-infrared window (NIR-II window). Due to its high spatial resolution of 22.79 µm, the NIR-II fluorescence imaging method could quickly distinguish the brain injury region of mice after performing the stab wound injury (traumatic brain injury) and ischemic stroke (non-traumatic brain injury), enabling it a powerful tool in the noninvasive and early diagnosis of ABI.

Photodynamic therapy reduces metastasis of breast cancer by minimizing circulating tumor cells.

Cancer metastasis after traditional surgery introduces a high barrier to therapy efficacy. Photodynamic therapy (PDT) for cancer is based on a photochemical process of photosensitizers that concentrate in tumors and release oxidant species under light excitation to destroy cells. Compared with traditional surgery, PDT provides minimal invasion and targeted therapy. In this in vivo study, we monitor the real-time and long-term dynamics of circulating tumor cells (CTCs) after a single round of PDT and after surgical resection in a breast cancer animal model. The CTC level is low after PDT treatment, and the recurrence of the primary tumor is postponed in the PDT group compared with the resection group. We find that metastasis is correlated with the CTC level, and the PDT-treated mice show no metastasis in the lung or liver. Our results suggest PDT can effectively reduce metastasis by minimizing CTCs after treatment and is a great technology for breast cancer therapy.

Ratiometric Raman nanotags enable intraoperative detection of metastatic sentinel lymph node.

Sentinel lymph node (SLN) imaging and biopsy has been advocated as an important technique to evaluate the metastatic status of regional lymph nodes and determine subsequent surgical procedure for many cancers, yet there is no reliable means to provide accurate and rapid diagnosis of metastatic SLN during surgery. Here we develop a new approach, named "Ratiometric Raman dual-nanotag strategy", that using folic acid functionalized targeted and nontargeted gap-enhanced Raman tags (FA-GERTs and Nt-GERTs) to detect metastatic SLN based on Raman imaging combined with classical least square data processing methods. By using this strategy, with built-in self-calibration for signal correction, rather than absolute intensity-dependent signal readout, we realize the visualization and prompt intraoperative diagnosis of metastatic SLN with a high accuracy of 87.5 % when the cut-off value of ratio (FA-GERTs/Nt-GERTs) set at 1.255. This approach may outperform the existing histopathological assessment in diagnosing SLN metastasis and is promising for guiding surgical procedure in the future.

Fundamentals of Optical Imaging.

Optical imaging, which possesses noninvasive and high-resolution features for biomedical imaging, has been used to study various biological samples, from in vitro cells, ex vivo tissue, to in vivo imaging of living organism. Furthermore, optical imaging also covers a very wide scope of spatial scale, from submicron sized organelles to macro-scale live biological samples, enabling it a powerful tool for biomedical studies. Before introducing these superior optical imaging methods to researchers, first of all, it is necessary to present the basic concept of light-matter interactions such as absorption, scattering, and fluorescence, which can be used as the imaging contrast and also affect the imaging quality. And then the working mechanism of various imaging modalities including fluorescence microscopy, confocal microscopy, multiphoton microscopy, super-resolution microscopy, optical coherence tomography (OCT), diffuse optical tomography (DOT), etc. will be presented. Meanwhile, the main features and typical bioimaging applications of these optical imaging technologies are discussed. Finally, the perspective of future optical imaging methods is presented. The aim of this chapter is to introduce the background and principle of optical imaging for grasping the mechanism of advanced optical imaging modalities introduced in the following chapters.

In Vivo Flow Cytometry.

In vivo flow cytometry (IVFC) was first designed to detect circulating cells in a mouse ear. It allows real-time monitoring of cells in peripheral blood with no need to draw blood. The IVFC field has made great progress during the last decade with the development of fluorescence, photoacoustic, and multiphoton microscopy. Moreover, the application of IVFC is no longer restricted to circulating cells. IVFC based on fluorescence and photoacoustic are most widely applied in biomedical research. Methods based on fluorescence are often used for object monitoring in superficial vessels, while methods based on photoacoustics have an advantage of label-free monitoring in deep vessels. In this chapter, we introduce technical points and key applications of IVFC. We focus on the principles, labeling strategies, sensitivity, and biomedical applications of the technology. In addition, we summarize this chapter and discuss important research directions of IVFC in the future.

Noninvasive and real-time monitoring of Au nanoparticle promoted cancer metastasis using in vivo flow cytometry.

Cancer is the second leading cause of mortality globally, while cancer metastasis, which accounts for about 90% of cancer-related mortality, presents an extremely poor prognosis. Thus, various nanomedicines were designed and synthesized for cancer treatment, but nanomaterials could lead to endothelial leakiness and consequently facilitate intravasation and extravasation of cancer cells to form circulating tumor cells (CTCs), which were regarded as the potential metastatic seeds, possibly accelerating cancer metastasis. Neither possible metastatic sites were observed nor rare CTCs could be measured using common methods at the early stage of cancer metastasis, it is urgent to explore new technology to dynamically monitor nanomedicine promoted cancer metastasis with high sensitivity, which would be beneficial for cancer treatment as well as design and synthesis of nanomedicine. Herein, a novel optical biopsy tool i.e. in vivo flow cytometry (IVFC) was constructed to noninvasively and real-time monitor CTCs of tumor-bearing mice treated with various concentrations of Au nanoparticles. The in vivo experimental results demonstrated the promoted CTCs were Au nanoparticles dose-dependent consistent with the in vitro results, which showed Au nanoparticles induced dose-dependent gaps in the blood vessel endothelial walls to accelerate CTCs formation, making IVFC a promising biopsy tool in fundamental, pre-clinical and clinical investigation of nanomedicine and cancer metastasis.

Rapid ex vivo assessment of cancer prognosis by fluorescence imaging of nucleolus using nitrogen doped carbon dots.

Cancer severely threatens human health currently, promoting the rapid development of cancer treatment strategies. In addition to cancer therapy, assessment of cancer prognosis, which can evaluate the success with treatment and chances of recovery as well as assist to make subsequent therapeutic schedule, is also remarkably indispensable and important. Conventional technologies can't provide rapid and highly-sensitive assessment of cancer prognosis at cytological level. Herein, an effective nitrogen doped carbon dots with intrinsic nucleolus-targeting capability and high fluorescence quantum yield are synthesized, characterized and employed for fluorescence imaging of nucleolus, which is closely related to the biological alteration of cancer cell. The cancer prognosis thus can be accurately (limit of detection: 50 nM) and rapidly (5 min) assessed at subcellular organelle level from nucleolar characteristics, which are visualized and analyzed by the captured fluorescence images. Outstanding assessment performance endows the proposed technology with great potential for future clinical research.

Near-infrared light excited photodynamic anticancer therapy based on UCNP@AIEgen nanocomposite.

Photodynamic therapy (PDT), a clinically approved cancer treatment strategy, features non-invasiveness, few side-effects, high spatial resolution, etc. The advancement of PDT has been significantly restricted by the penetration depth of the excitation light. Herein, an effective fluorogen, TBD, with aggregation-induced emission characteristics (AIEgen) and high reactive-oxygen-species (ROS) generation efficiency was reported and integrated with a near infrared (NIR) light excitable upconversion nanoparticle (UCNP) to construct NIR light excitable UCNP@TBD nanocomposites. The formed nanocomposite has excellent photostability, good biocompatibility, and efficient ROS generation under NIR light excitation via Förster resonance energy transfer (FRET), enabling NIR light excited PDT. Moreover, the proposed NIR light excited PDT can break the impasse between the penetration depth and excitation volume in conventional PDT, effectively improving the anticancer therapeutic outcome. In vitro cancer cell ablation and in vivo tumor growth inhibition validated that the proposed UCNP@TBD nanocomposite is a promising NIR light excitable PDT agent with great potential for future translational research.

Nucleolus-Targeted Photodynamic Anticancer Therapy Using Renal-Clearable Carbon Dots.

Photodynamic therapy (PDT), which utilizes light excited photosensitizers (PSs) to generate reactive oxygen species (ROS) and consequently ablate cancer cells or diseased tissue, has attracted a great deal of attention in the last decades due to its unique advantages. In order to further enhance PDT effect, PSs are functionalized to target specific sub-cellular organelles, but most PSs cannot target nucleolus, which is demonstrated as a more efficient and ideal site for cancer treatment. Here, an effective carbon dots (C-dots) photosensitizer with intrinsic nucleolus-targeting capability, for the first time, is synthesized, characterized, and employed for in vitro and in vivo image-guided photodynamic anticancer therapy with enhanced treatment performance at a low dose of PS and light irradiation. The C-dots possess high ROS generation efficiency and fluorescence quantum yield, excellent in vitro and in vivo biocompatibility, and rapid renal clearance, endowing it with a great potential for future translational research.

Flexible porphyrin doped polymer optical fibers for rapid and remote detection of trace DNT vapor.

With the rapid growth of anti-terrorist activities worldwide, it becomes an emerging requirement to rapidly and accurately detect hidden explosive threats. However, the safety issue during the explosive material detection, e.g. unexpected explosion, is still an insurmountable challenge. In this study, we design and mass-produce a novel kind of flexible 5,10,15,20-tetrakis(4-aminophenyl)porphyrin doped polymer optical fiber (PPOF) for rapid and accurate detection of trace 2,4-dinitrotoluene (DNT) vapor based on the DNT induced florescence quenching mechanism. The influence of doping concentration, bending, and temperature on the sensing performance is investigated. PPOF shows immunity to bending, enabling it to work in a harsh environment. It is experimentally demonstrated that the limit of detection and response time of the proposed PPOF could reach around 120 ppb and 3 minutes, respectively, which make it much better than other techniques. Owning to its inherent advantages including low-cost, remote-control capability, and compatibility with optical communication networking, PPOF can be constructed the quasi-distributed sensing networking of explosive matters in the future, providing a new strategy for anti-terrorism.

Recent advances in copper sulphide-based nanoheterostructures.

Due to the high mobility of copper ions in numerous structurally-related phases, copper sulphide (Cu2-xS, 0 ≤x≤ 1) has been widely used as a starting template to fabricate various heterostructures via cation exchange. Such nanoheterostructures can possess unique combinations of physical properties that could be useful in diverse applications. Controllable methods of fabricating copper sulphide nanoheterostructures of increasing complexity have been rapidly emerging over the past few years. In this tutorial review, we discuss recent progress in heterostructure fabrication methods using copper sulphide. We primarily focus on important reports of cation exchange-based approaches and then summarize some key emerging applications that can employ these copper-sulphide-based nanoheterostructures.

In vitro anticancer activity of AIEgens.

Fluorogens with aggregation-induced emission (AIE) characteristics (AIEgens) possess unique optical properties, design flexibility, and multi-functional capabilities and have established their niche as smart materials since their discovery in 2001. In recent years, AIEgens have found varied applications in sensing, imaging, and therapy in biomedical research. In this work, we systematically and comprehensively investigate the in vitro anticancer activity of AIEgens. We report the high cytotoxicity of AIEgens against cancer cells, especially against cancer stem cells (CSCs) which show high resistance to existing therapeutic drug regimens. Furthermore, we explore the role of AIEgens as novel image-guided chemotherapy agents that offer a new avenue for efficient cancer treatment.