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Tumor vaccine therapy offers significant advantages over conventional treatments, including reduced toxic side effects. However, it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods. Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors, thereby limiting their clinical efficacy. In this study, we developed a nanoparticle-based tumor vaccine, OVA@HA-PEI, utilizing ovalbumin (OVA) as the presenting antigen and hyaluronic acid (HA) and polyethyleneimine (PEI) as adjuvants and carriers. This formulation significantly enhanced the proliferation of immune cells and cytokines, such as CD3, CD8, interferon-[Formula: see text], and tumor necrosis factor-[Formula: see text], in vivo, effectively activating an immune response against B16–F10 tumors. In vivo fluorescence flow cytometry (IVFC) has already become an effective method for monitoring circulating tumor cells (CTCs) due to its direct, noninvasive, and long-term detection capabilities. Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1 (PD-1) antibody. The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone. Additionally, this combination therapy substantially reduced the number of CTCs in vivo, increased the clearance rate of CTCs by the immune system, and slowed tumor progression. These findings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Abstract Sperm quality analysis plays an important role in diagnosing infertility, which is widely implemented by computer‐assisted sperm analysis (CASA) of sperm‐swimming imaging from commercial phase‐contrast microscopy. A well‐equipped microscope comes with a high cost, increasing the burden of assessment, and it also occupies a large volume. For point‐of‐care testing (POCT) of sperm quality, these factors are confronted with the challenges of low‐cost and portable instruments. In this study, an encoded light‐emitting diode (LED) array illumination is employed to achieve a portable microscope with multicontrast imaging for sperm quality analysis. This microscopy has dimensions of 16.5 × 14.0 × 25.0 cm, and its dark‐field (DF) imaging provides high‐contrast sperm image data which is suitable for CASA. According to DF imaging, we developed a software of LabCASA, which can used to assess the motility characteristics of sperm. Compared with TrackMate, the difference in motility parameters from our software was less than 10% in the coefficient of variation (CV). The sperm motility parameters vary with the chamber temperature, which further confirms the reliability of our system with DF imaging. The DF imaging provides strong robustness for tracking sperm's motion under different microscopes. For assessment of the motility parameters, our system can work at a lower cost with a plastic structure. This system with DF imaging is suitable for portable POCT of sperm quality analysis, which is highly cost‐effective in resource‐constrained circumstances.

Abstract Fluorogens with aggregation‐induced emission (AIEgens) are promising agents for two‐photon fluorescence (TPF) imaging. However, AIEgens’ photophysical properties are fixed and unoptimizable once synthesized. Therefore, it is urgent and meaningful to explore an efficient post‐regulation strategy to optimize AIEgens’ photophysical properties. Herein, a general and efficient post‐regulation strategy is reported. By simply tuning the ratio of inert AIEgens within binary nanoparticles (BNPs), the fluorescence quantum yield and two‐photon absorption cross‐section of functional AIEgens are enhanced by 8.7 and 5.4 times respectively, which are not achievable by conventional strategies, and the notorious phototoxicity is almost eliminated. The experimental results, theoretical simulation, and mechanism analysis demonstrated its feasibility and generality. The BNPs enabled deep cerebrovascular network imaging with ≈1.10 mm depth and metastatic cancer cell detection with single‐cell resolution. Furthermore, the TPF imaging quality is improved by the self‐supervised denoising algorithm. The proposed binary molecular post‐regulation strategy opened a new avenue to efficiently boost the AIEgens’ photophysical properties and consequently TPF imaging quality.

Abstract Extracellular vesicles (EVs) are vesicular structures comprised of a bilayer lipid membrane, released by living cells. There is a growing body of evidence for their functionality, indicating that small EVs (sEVs) can mediate specific forms of intercellular communication. The future applications of sEVs hold great promise, not only as diagnostic markers but also as therapeutic agents. However, the greatest difficulty in the clinical translation of sEVs is that they are currently poorly understood, especially concerning their in vivo behaviour. In this study, we provide a novel method for monitoring sEVs in blood circulation based on in vivo flow cytometry (IVFC). We have demonstrated that the height of the IVFC signal baseline is proportional to the concentration of sEVs. Moreover, we have found out that the peaks in the IVFC signal are generated by the aggregation or cellular uptake of sEVs. In vivo monitoring of sEVs clearance from the blood indicates that PEGylated sEVs have a longer residence time and exhibit less aggregation in circulation compared to non‐PEGylated sEVs. These studies reveal that IVFC enables real‐time in vivo monitoring of circulating sEVs, which can provide valuable insights into the pharmacokinetics and cellular targeting capabilities of sEVs.

Melanoma is a highly metastatic and lethal skin tumor originating from melanocyte malignancy. Circulating tumor cells (CTCs) are key endogenous biomarkers in melanoma metastasis. Melanin and blood vessels exhibit substantial disparities in their absorbance profiles at select wavelengths, a characteristic that can be adeptly harnessed to differentiate the photoacoustic signals they generate. Photoacoustic flow cytometry (PAFC), which harnesses this principle, enables the monitoring of CTC flowing in vivo. However, this technique is constrained by the inefficiency and high false positive rates associated with traditional algorithms, including the Pauta criterion. In this study, a PAFC system is developed to identify dynamic features of flowing CTCs and the inhibitory effects of PD-L1 inhibitors, using a one-dimensional convolutional neural network (1D-CNN) and a long short-term memory (LSTM) network. The 1D-CNN achieves a balance between classification accuracy and speed. Meanwhile, the LSTM exhibits superior specificity but limited sensitivity. By combining the advantages of the two networks, the inhibitory effect of PD-L1 inhibitors that reduce the CTCs in the blood and block metastasis to other organs of melanoma mouse models are studied noninvasively in vivo and validated in vitro. The PAFC equipped with the deep learning framework provides a more timely and efficient assessment of PD-L1 inhibitors compared to conventional pathological methods, significantly enhancing the melanoma diagnosis and treatment monitoring. This technology demonstrates potential as a significant tool for the non-invasive, dynamic evaluation of melanoma progression and response to immunotherapy.

In-vivo flow cytometry is a noninvasive real-time diagnostic technique that facilitates continuous monitoring of cells without perturbing their natural biological environment, which renders it a valuable tool for both scientific research and clinical applications. However, the conventional approach for improving classification accuracy often involves labeling cells with fluorescence, which can lead to potential phototoxicity. This study proposes a label-free in-vivo flow cytometry technique, called dynamic YOLOv4 (D-YOLOv4), which improves classification accuracy by integrating absorption intensity fluctuation modulation (AIFM) into YOLOv4 to demodulate the temporal features of moving red blood cells (RBCs) and platelets. Using zebrafish as an experimental model, the D-YOLOv4 method achieved average precisions (APs) of 0.90 for RBCs and 0.64 for thrombocytes (similar to platelets in mammals), resulting in an overall AP of 0.77. These scores notably surpass those attained by alternative network models, thereby demonstrating that the combination of physical models with neural networks provides an innovative approach toward developing label-free in-vivo flow cytometry, which holds promise for diverse in-vivo cell classification applications.

Abstract Background We tested the hypothesis that radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) promotes tumor cell release and explored a method for reducing these effects. Methods A green fluorescent protein-transfected orthotopic HCC model was established in 99 nude mice. In vivo flow cytometry was used to monitor circulating tumor cell (CTC) dynamics. Pulmonary fluorescence imaging and pathology were performed to investigate lung metastases. First, the kinetics of CTCs during the periablation period and the survival rate of CTCs released during RFA were investigated. Next, mice were allocated to controls, sham ablation, or RFA with/without hepatic vessel blocking (ligation of the portal triads) for evaluating the postablation CTC level, lung metastases, and survival over time. Moreover, the kinetics of CTCs, lung metastases, and mice survival were evaluated for RFA with/without ethanol injection. Pathological changes in tumors and surrounding parenchyma after ethanol injection were noted. Statistical analysis included t-test, ANOVA, and Kaplan-Meier survival curves. Results CTC counts were 12.3-fold increased during RFA, and 73.7% of RFA-induced CTCs were viable. Pre-RFA hepatic vessel blocking prevented the increase of peripheral CTCs, reduced the number of lung metastases, and prolonged survival (all p ≤ 0.05). Similarly, pre-RFA ethanol injection remarkably decreased CTC release during RFA and further decreased lung metastases with extended survival (all p ≤ 0.05). Histopathology revealed thrombus formation in blood vessels after ethanol injection, which may clog tumor cell dissemination during RFA. Conclusion RFA induces viable tumor cell dissemination, and pre-RFA ethanol injection may provide a prophylactic strategy to reduce this underestimated effect. Relevance statement RFA for HCC promotes viable tumor cell release during ablation, while ethanol injection can prevent RFA induced tumor cell release. Key points • RFA induced the release of viable tumor cells during the ablation procedure in an animal model. • Hepatic vessel blocking can suppress tumor cells dissemination during RFA. • Ethanol injection can prevent RFA-induced tumor cell release, presumably because of the formation of thrombosis. Graphical Abstract

Abstract Chemotherapy remains as the first-choice treatment option for triple-negative breast cancer (TNBC). However, the limited tumor penetration and low cellular internalization efficiency of current nanocarrier-based systems impede the access of anticancer drugs to TNBC with dense stroma and thereby greatly restricts clinical therapeutic efficacy, especially for TNBC bone metastasis. In this work, biomimetic head/hollow tail nanorobots were designed through a site-selective superassembly strategy. We show that nanorobots enable efficient remodeling of the dense tumor stromal microenvironments (TSM) for deep tumor penetration. Furthermore, the self-movement ability and spiky head markedly promote interfacial cellular uptake efficacy, transvascular extravasation, and intratumoral penetration. These nanorobots, which integrate deep tumor penetration, active cellular internalization, near-infrared (NIR) light-responsive release, and photothermal therapy capacities into a single nanodevice efficiently suppress tumor growth in a bone metastasis female mouse model of TNBC and also demonstrate potent antitumor efficacy in three different subcutaneous tumor models.

Abstract Light therapy techniques, such as photobiomodulation therapy (PBMT), photodynamic therapy (PDT), and laser photoablation, have gained widespread attention and become indispensable physiotherapy methods in clinical practice. PBMT involves the application of low‐level laser/LED to modulate the function of nerve cells, relieve neuroinflammation, promote neurogenesis and vascular growth. Recent studies have shown that PBMT holds promise as a complementary or alternative treatment of Alzheimer's disease (AD), traumatic brain injury (TBI), major depressive disorder (MDD), etc. However, the therapeutic effect of PBMT is influenced by various factors, such as the patients' condition, brain structure and function, illumination parameters, etc. Therefore, the optimized parameters, personalized therapeutic schedules, and precise evaluation of the therapeutic effect are crucial to the treatment success. In this review, we identified the recent experimental and clinical successes, existing obstacles, and future opportunities for PBMT in the treatment of the brain diseases. As a non‐invasive, side‐effect‐free, and highly accessible technique, PBMT brings a glimmer of light for the treatment of neuropsychiatric disorders and the neuro‐rejuvenation of human brains.

Abstract Malignant breast cancer (BC) remains incurable mainly due to the cancer cell metastasis, which is mostly related to the status of Estrogen receptor alpha (ERα). However, our understanding of the mechanisms through which ERα regulates cancer cell metastasis remains limited. Here we identified a miR-29a-PTEN-AKT axis as a downstream signaling pathway of ERα governing breast cancer progression and metastasis. Two estrogen response element (ERE) half sites were identified in the promoter and enhancer regions of miR-29a, which mediated transcriptional regulation of miR-29a by ERα. Low level of miR-29a showed association with reduced metastasis and better survival in ERα+ luminal subtype of BC. In contrast, high level of miR-29a was detected in ERα- triple negative breast cancer (TNBC) in association with distant metastasis and poor survival. miR-29a overexpression in BC tumors increased the number of circulating tumor cells and promoted lung metastasis in mice. Targeted knockdown of miR-29a in TNBC cells in vitro or administration of a nanotechnology-based anti-miR-29a delivery in TNBC tumor-bearing mice in vivo suppressed cellular invasion, EMT and lung metastasis. PTEN was identified as a direct target of miR-29a, inducing EMT and metastasis via AKT signaling. A small molecular inhibitor of AKT attenuated miR-29a-induced EMT. These findings demonstrate a novel mechanism responsible for ERα-regulated breast cancer metastasis, and reveal the combination of ERα status and miR-29a levels as a new risk indicator in BC.

Clinical measurement of corneal biomechanics can aid in the early diagnosis, progression tracking, and treatment evaluation of ocular diseases. Over the past two decades, interdisciplinary collaborations between investigators in optical engineering, analytical biomechanical modeling, and clinical research has expanded our knowledge of corneal biomechanics. These advances have led to innovations in testing methods (ex vivo, and recently, in vivo) across multiple spatial and strain scales. However, in vivo measurement of corneal biomechanics remains a long-standing challenge and is currently an active area of research. Here, we review the existing and emerging approaches for in vivo corneal biomechanics evaluation, which include corneal applanation methods, such as ocular response analyzer (ORA) and corneal visualization Scheimpflug technology (Corvis ST), Brillouin microscopy, and elastography methods, and the emerging field of optical coherence elastography (OCE). We describe the fundamental concepts, analytical methods, and current clinical status for each of these methods. Finally, we discuss open questions for the current state of in vivo biomechanics assessment techniques and requirements for wider use that will further broaden our understanding of corneal biomechanics for the detection and management of ocular diseases, and improve the safety and efficacy of future clinical practice.

Platelets buoy up cancer metastasis via arresting cancer cells, enhancing their adhesion, and facilitating their extravasation through the vasculature. When deprived of intracellular and granular contents, platelet decoys could prevent metastatic tumor formation. Inspired by these, we developed nanoplatesomes by fusing platelet membranes with lipid membranes (P-Lipo) to restrain metastatic tumor formation more efficiently. It was shown nanoplateletsomes bound with circulating tumor cells (CTC) efficiently, interfered with CTC arrest by vessel endothelial cells, CTC extravasation through endothelial layers, and epithelial-mesenchymal transition of tumor cells as nanodecoys. More importantly, in the mouse breast tumor metastasis model, nanoplateletsomes could decrease CTC survival in the blood and counteract metastatic tumor growth efficiently by inhibiting the inflammation and suppressing CTC escape. Therefore, nanoplatelesomes might usher in a new avenue to suppress lung metastasis.

The study of circulating cells in the blood stream is critical, as it covers many fields of biomedicine, including immunology, cell biology, oncology, and reproductive medicine. In-vivo flow cytometry (IVFC) is a new tool to monitor and count cells in real time for long durations in their native biological environment. This review describes two main categories of IVFC, i.e., labeled and label-free IVFC. It focuses on label-free IVFC and introduces its technological development and related biological applications. Because cell recognition is the basis of flow cytometry counting, this review also describes various methods for the classification of unlabeled cells, including the latest machine learning-based technologies.

The skin is the largest organ in humans. It comprises about 16% of our body. Many diseases originate from the skin, including acne vulgaris, skin cancer, fungal skin disease, etc. As a common skin cancer in China, melanoma alone grows at year rate of nearly 4%. Therefore, it is crucial to develop an objective, reliable, accurate, non-invasive, and easy-to-use diagnostic method for skin diseases to support clinical decision-making. Raman spectroscopy is a highly specific imaging technique, which is sensitive, even to the single-cell level in skin diagnosis. Raman spectroscopy provides a pattern of signals with narrow bandwidths, making it a common and essential tool for researching individual characteristics of skin cells. Raman spectroscopy already has a number of clinical applications, including in thyroid, cervical and colorectal cancer. This review will introduce the advantages and recent developments in Raman spectroscopy, before focusing on the advances in skin diagnosis, including the advantages, methods, results, analysis, and notifications. Finally, we discuss the current limitations and future progress of Raman spectroscopy in the context of skin diagnosis.

We reported a newly engineered photosensitizer of polymer encapsulated carbonized hemin nanoparticles (P-CHNPs) via a facile synthesis procedure for boosting photodynamic therapy (PDT). The synthesized P-CHNPs achieved enhanced oxidative stress in tumor microenvironment, which could be further amplified under light irradiation, enabling excellent in vitro and in vivo PDT effects. 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.

Nanotheranostic agents that integrate diagnosis and treatment are promising for precision medicine, but they encounter some obstacles such as penetration depth and efficiency. In this study, novel carbon nitride-rose bengal nanoparticles (CN-RB NPs) with a graphite carbon nitride skeleton were synthesized by one-step thermal copolymerization. The enhanced absorption in the near-infrared-II region (NIR-II) endows CN-RB NPs with an excellent photothermal effect under 1064 nm laser irradiation, as well as an obvious photoacoustic signal for imaging in vivo. Interestingly, due to the introduced iodine element, CN-RB NPs exhibit enhanced radiation therapy, indicating that CN-RB NPs can achieve ideal therapeutic outcome through collaborative photothermal/radiation therapy under the guidance of NIR-II photoacoustic imaging. Moreover, CN-RB NPs demonstrate minimal side effects and long-term biological stability after 14 days. Therefore, the proposed new multifunctional nano-platform CN-RB NPs hold great potential in the application of deep therapeutics.

This special issue collects 20 excellent papers, spanning NIR II imaging, high-speed imaging, adaptive wavefront shaping, label-free imaging, ultrasensitive detection, polarization optics, photodynamic therapy, and preclinical applications.

Abstract Photobiomodulation, by utilizing low-power light in the visible and near-infrared spectra to trigger biological responses in cells and tissues, has been considered as a possible therapeutic strategy for Alzheimer’s disease (AD), while its specific mechanisms have remained elusive. Here, we demonstrate that cognitive and memory impairment in an AD mouse model can be ameliorated by 1070-nm light via reducing cerebral β-amyloid (Aβ) burden, the hallmark of AD. The glial cells, including microglia and astrocytes, play important roles in Aβ clearance. Our results show that 1070-nm light pulsed at 10 Hz triggers microglia rather than astrocyte responses in AD mice. The 1070-nm light-induced microglia responses with alteration in morphology and increased colocalization with Aβ are sufficient to reduce Aβ load in AD mice. Moreover, 1070-nm light pulsed at 10 Hz can reduce perivascular microglia and promote angiogenesis to further enhance Aβ clearance. Our study confirms the important roles of microglia and cerebral vessels in the use of 1070-nm light for the treatment of AD mice and provides a framework for developing a novel therapeutic approach for AD.

Abstract Refractive surgery is recognized as an effective method for myopia treatment, but it can induce night vision disturbances such as glare. We present an eye modeling method for the optical quality assessment in response to the structural changes in the eyes by femto-LASIK surgery. Customized eye models were built from the measurements of 134 right eyes pre- and post-operatively. Optical performance was evaluated using spot diagrams, point spread functions (PSFs), modulation transfer functions (MTFs), and chromatic aberrations at various fields (0°–30°), different pupil diameters (2–6 mm), and initial myopias (− 1.25 to − 10.5 D). Pupil size and initial myopia are the two major factors that affect visual performance of post-operative eyes. The results of spot diagrams, PSFs, and MTFs indicated that post-operative visual performance deteriorated as the visual field and pupil size increased, and it was significantly influenced by initial myopia. Post-operative chromatic aberrations were also affected by initial myopia. As pupil size increased, the post-operative longitudinal chromatic aberrations tended to decrease slightly, while the transverse chromatic aberrations remained similar. The use of eye modeling for refractive surgery assessment could possibly provide a more personalized surgical approach, could improve the prediction accuracy of refractive surgery outcomes, and promote the invention and development of better surgical methods.

Abstract Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

We propose a novel retinal layer segmentation method to accurately segment 10 retinal layers in optical coherence tomography (OCT) images with intraretinal fluid. The method used a fan filter to enhance the linear information pertaining to retinal boundaries in an OCT image by reducing the effect of vessel shadows and fluid regions. A random forest classifier was employed to predict the location of the boundaries. Two novel methods of boundary redirection (SR) and similarity correction (SC) were combined to carry out boundary tracking and thereby accurately locate retinal layer boundaries. Experiments were performed on healthy controls and subjects with diabetic macular edema (DME). The proposed method required an average of 415[Formula: see text]s for healthy controls and of 482[Formula: see text]s for subjects with DME and achieved high accuracy for both groups of subjects. The proposed method requires a shorter running time than previous methods and also provides high accuracy. Thus, the proposed method may be a better choice for small training datasets.

The gold standard for diagnosis and monitoring of myocardial infarction is a chemiluminescence assay based on the detection of cardian troponin I (cTnI). Here, the authors develop a plasmonic gold nano-island chip assay for ultrasensitive detection of cTnI in as little as 10 μL of serum.

Photodynamic therapy (PDT) takes advantage of photosensitizers (PSs) to generate reactive oxygen species (ROS) for cell killing when excited by light. It has been widely used in clinic for therapy of multiple cancers. Currently, all the FDA-approved PSs, including porphyrin, are all small organic molecules, suffering from aggregation-caused quenching (ACQ) issues in biological environment and lacking tumor targeting capability. Nanoparticles (NPs) with size between 20nm and 200nm possess tumor targeting capability due to the enhanced permeability and retention (EPR) effect. It is urgent to develop a new strategy to form clinical-approved-PSs-based NPs with improved ROS generation capability. In this study, we report a strategy to overwhelm the ACQ of porphyrin by doping it with a type of aggregation-induced emission (AIE) luminogen to produce a binary NPs with high biocompatibility, and enhanced fluorescence and ROS generation capability. Such NPs can be readily synthesized by mixing a porphyrin derivative, Ce6 with a typical AIE luminogen, TPE-Br. Here, our experimental results have demonstrated the feasibility and effectiveness of this strategy, endowing it a great potential in clinical applications.

Rapid detection of foodborne pathogens is one of the most effective ways to solve food safety problems. To achieve rapid and noninvasive detection and classification of foodborne pathogens, we modified a fiber confocal backscattering micro-spectral system to suit an extremely small biological sample, that is, a bacterium. This system offers single-bacterium level, label-free, convenient, and environmentally friendly characterization. Three categories of common foodborne pathogens (Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus) were measured. The scattering spectrum ranging from 450 to 900 nm was selected, and by the model of principal component analysis (PCA) and error back propagation algorithm of back propagation neural network (BPNN), the backscattering microscopic spectra of three categories of pathogens were dimensionally reduced, identified, and classified. The results showed that the identification accuracy of three categories of pathogens was above 90%, under neutral, acidic, and alkaline culturing conditions, respectively. The preliminary results demonstrated the feasibility of using confocal backscattering microscopic spectra combined with PCA and BPNN algorithm to identify and classify single bacterium in a rapid, noninvasive, and label-free manner.

Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.

The fluorescence-based in vivo flow cytometry (IVFC) is an emerging tool to monitor circulating cells in vivo. As a noninvasive and real-time diagnostic technology, the fluorescence-based IVFC allows long-term monitoring of circulating cells without changing their native biological environment. It has been applied for various biological applications (e.g., monitoring circulating tumor cells). In this work, we will review our recent works on fluorescence-based IVFC. The operation principle and typical biological applications will be introduced. In addition, the recent advances in IVFC flow cytometry based on photoacoustic effects and other label-free detection methods such as imaging-based methods, diffuse-light methods, hybrid multimodality methods and multispectral methods are also summarized.

Recent studies in oncology have addressed the importance of detecting circulating tumor cell clusters because circulating tumor cell clusters might survive and metastasize more easily than single circulating tumor cells. Signals with larger peak widths detected by in vivo flow cytometer (IVFC) have been used to identify cell clusters in previous studies. However, the accuracy of this criterion might be greatly degraded by variance in blood flow and the rolling behaviors of circulating tumor cells. Here, we propose a criterion and algorithm to distinguish cell clusters from single cells. In this work, we first used area-based and volume-based models for single fluorescent cells. Simulating each model, we analyzed the corresponding morphology of IVFC signals from cell clusters. According to the Rayleigh criterion, the valley between two adjacent peak signals from two distinguishable cells should be lower than 73.5% of the peak values. A novel signal processing algorithm for IVFC was developed based on this criterion. The results showed that cell clusters can be reliably identified using our proposed algorithm. Intravital imaging was also performed to further support our algorithm. With enhanced accuracy, IVFC is a powerful tool to study circulating cell clusters.

Two-photon microscopy (TPM) has been one of the most widely used fluorescent microscopy methods in biological tissue. The femtosecond laser in the near-infrared (NIR) range showed better penetration in tissue due to less scattering and absorption. The method to estimate the optimal wavelength generally according to the scattering and absorption is somehow too rough considering the complex biological property and other factors that influence TPM. In this report, we set up a model to calculate the optimal wavelength in the NIR range for different imaging depths by involving more parameters of TPM. The calculated result suggests the image of brain slice stained with fluorescein isothiocyanate (FITC) can be acquired with the best TPM imaging quality at 780 nm excitation rather than longer wavelengths, which is consistent with our experimental results of TPM at depth of 50-300 μm in mouse brain tissue. Our results may, therefore, bring a better understanding on TPM in tissue and help to develop a more accurate model for the simulation of TPM.

T-cell activation requires the formation of the immunological synapse (IS) between a T-cell and an antigen-presenting cell (APC) to control the development of the adaptive immune response. However, calcium release, an initial signal of T-cell activation, has been found to occur before IS formation. The mechanism for triggering the calcium signaling and relationship between calcium release and IS formation remains unclear. Herein, using live-cell imaging, we found that intercellular adhesion molecule 1 (ICAM-1), an essential molecule for IS formation, accumulated and then was depleted at the center of the synapse before complete IS formation. During the process of ICAM-1 depletion, calcium was released. If ICAM-1 failed to be depleted from the center of the synapse, the sustained calcium signaling could not be induced. Moreover, depletion of ICAM-1 in ISs preferentially occurred with the contact of antigen-specific T-cells and dendritic cells (DCs). Blocking the binding of ICAM-1 and lymphocyte function-associated antigen 1 (LFA-1), ICAM-1 failed to deplete at the center of the synapse, and calcium release in T-cells decreased. In studying the mechanism of how the depletion of ICAM-1 could influence calcium release in T-cells, we found that the movement of ICAM-1 was associated with the localization of LFA-1 in the IS, which affected the localization of calcium microdomains, ORAI1 and mitochondria in IS. Therefore, the depletion of ICAM-1 in the center of the synapse is an important factor for an initial sustained calcium release in T-cells.

In the traditional two-step procedure used in intensity-modulated radiation therapy, fluence map optimization (FMO) is performed first, followed by use of a leaf sequencing algorithm (LSA). By contrast, direct aperture optimization (DAO) directly optimizes aperture leaf positions and weights. However, dose calculation using the Monte Carlo (MC) method for DAO is often time-consuming. Therefore, a rapid DAO (RDAO) algorithm is proposed that uses a dose influence matrix based piecewise aperture dose model (DIM-PADM). In the proposed RDAO algorithm, dose calculation is based on the dose influence matrix instead of MC. The dose dependence of aperture leafs is modeled as a piecewise function using the DIM. The corresponding DIM-PADM-based DAO problem is solved using a simulated annealing algorithm.The proposed algorithm was validated through application to TG119, prostate, liver, and head and neck (H&N) cases from the common optimization for radiation therapy dataset. Compared with the two-step FMO-LSA procedure, the proposed algorithm resulted in more precise dose conformality in all four cases. Specifically, for the H&N dataset, the cost value for the planned target volume (PTV) was decreased by 32%, whereas the cost value for the two organs at risk (OARs) was decreased by 60% and 92%. Our study of the proposed novel DIM-PADM-based RDAO algorithm makes two main contributions: First, we validate the use of the proposed algorithm, in contrast to the FMO-LSA framework, for direct optimization of aperture leaf positions and show that this method results in more precise dose conformality. Second, we demonstrate that compared to MC, the DIM-PADM-based method significantly reduces the computational time required for DAO.

Alzheimer’s disease (AD) is a chronic neurodegenerative disease. The symptoms include memory and spatial learning difficulties, language disorders, and loss of motivation, which get worse over time, eventually ending in death. No effective treatments are available for AD, currently. Current treatments only attenuate symptoms temporarily and are associated with severe side effects. Near infra-red (NIR) light has been studied for a long time. We investigated the effect of NIR on AD using a transgenic mouse model, which was obtained by co-injecting two vectors carrying AD mutations in amyloid precursor protein (APP) and presenilin-1 (PSEN1) into C57BL/6J mice. The irradiation equipment consisted of an accommodating box and an LED array. The wavelength of NIR light emitted from LED was between 1040nm and 1090nm. The power density delivered at the level of the mice was approximately 15mW/cm2. Firstly, we treated the mice with NIR for 40 days. Then, the irradiation was suspended for 28 days. Finally, another 15 days treatment was brought to mice. We conducted Morris water maze and immunofluorescence analysis to evaluate the effects of treatment. Immunofluorescence analysis was based on measuring the quantity of plaques in mouse brain slices. Our results show that NIR light improves memory and spatial learning ability and reduces plaques moderately. NIR light represents a potential treatment for AD.

In biomedical research fields, the in vivo flow cytometry (IVFC) is a widely used technology which is able to monitor target cells dynamically in living animals. Although the setup of IVFC system has been well established, baseline drift is still a challenge in the process of quantifying circulating cells. Previous methods, i.e., the dynamic peak picking method, counted cells by setting a static threshold without considering the baseline drift, leading to an inaccurate cell quantification. Here, we developed a method of cell counting for IVFC data with baseline drift by interpolation fitting, automatic segmentation and wavelet-based denoising. We demonstrated its performance for IVFC signals with three types of representative baseline drift. Compared with non-baseline-correction methods, this method showed a higher sensitivity and specificity, as well as a better result in the Pearson’s correlation coefficient and the mean-squared error (MSE).

Photodynamic therapy (PDT) has been commonly used in treating many diseases, such as cancer and infectious diseases. We investigated the different effects of PDT on three main pathogenic bacteria of periodontitis — Prevotella melaninogenica (P.m.), Porphyromonas gingivalis (P.g.) and Aggregatibacter actinomycetemcomitans (A.a.). The portable red light-emitting diode (LED) phototherapy device was used to assess the exogenous PDT effects with different light doses and photosensitizer concentrations (Toluidine blue O, TBO). The portable blue LED phototherapy device was used to assess the endogenous PDT effects with the use of endogenous photosensitizers (porphyrin) under different light doses. We found out that both exogenous and endogenous PDT were able to restrict the growth of all the three bacteria significantly. Moreover, the optimal PDT conditions for these bacteria were obtained through this in vitro screening and could guide the clinical PDT on periodontitis.

In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A(2A)) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A(2A) antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl(3)-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A(2A). Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.