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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 FROM AUTHOR]

Soluble CD83 (sCD83) exerts immunosuppressive functions in many autoimmune diseases, including experimental autoimmune uveitis (EAU), but the cells and mechanisms involved are unclear. This study showed that CD83+ B cells were the main sources of sCD83. They alleviated the symptoms of EAU and decreased the percentage of T cells and DCs in the eyes and lymph nodes. These CD83+ B cells decreased IL‐1β, IL‐18 and IFN‐γ secretion by DCs through sCD83. sCD83 interacted with GTPase Ras‐related protein (Rab1a) in DCs to promote Rab1a accumulation in autolysosomes and inhibit mTORC1 phosphorylation and NLRP3 expression. Hence, CD83+ B cells play a regulatory role in EAU by secreting sCD83. The lack of regulation of CD83+ B cells might be an important factor leading to hyperimmune activation in patients with autoimmune uveitis. CD83+ B cells suppress activated DCs in uveitis, indicating the potential therapeutic role of CD83+ B cells in uveitis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Nanoplateletsomes were inaugurated to restrain metastatic tumor formation efficiently through decoy and active targeting, serving as a platform to orchestrate the inhibition of circulating tumor cell survival and metastasis. [Display omitted] [ABSTRACT FROM AUTHOR]

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 s for healthy controls and of 482 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. [ABSTRACT FROM AUTHOR]

Author(s): Dorothy A. Sipkins (corresponding author) [1, 2, 3]; Xunbin Wei [1]; Juwell W. Wu [1]; Judith M. Runnels [1]; Daniel Côté [1]; Terry K. Means [4]; Andrew D. Luster [...]

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 20 nm and 200 nm 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. [ABSTRACT FROM AUTHOR]

Patient-derived organoids (PDOs) closely recapitulate human colorectal cancer biology and have recently emerged as preclinical models for personalized therapy design.1 However, PDOs cultured in Matrigel (PDOs SP Matrigel sp ) failed to predict outcome for treatment with the first-line FO chemotherapeutic regimen (5-fluorouracil plus oxaliplatin) for metastatic colorectal cancer (mCRC) patients.2 In this study, we establish in vitro culture conditions of mCRC-PDOs in a hydroxypropyl cellulose allyl conjugated with collagen (HA-Coll sponge),3 and utilized this system to examine the drug sensitivity of FO regimen (Figure 1A). PDOs in HA-Coll sponge (PDOs SP Sponge sp ) proliferated at the same level as PDOs SP Matrigel sp in the next generation (Figure 2B and C, Figure S1C and D). Conclusively, our results indicated that PDOs SP Sponge sp remained the original epithelial state of parental tumor tissues, which is important for the drug-sensitivity of FO, but not for irinotecan. To further confirm these results, we knocked out I LMNA i in PDOs SP Sponge sp and overexpressed I LMNA i in PDOs SP Matrigel sp to perform the drug response assay. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

We welcome the readers to the special issue on single cell analysis (Part II), which highlights recent progress in the development of single-cell optical analysis techniques and their applications in biological discovery, disease diagnosis, and treatment. Coherent Raman scattering (CRS) microscopy is capable of chemically selective, highly sensitive, submicron resolution, and high-speed imaging of lipid molecules in single live cells without any labeling. Raman spectroscopy is a highly specific imaging technique, which is sensitive, even to the single-cell level in skin diagnosis. [Extracted from the article]

Objective: Visualizing cell interactions in blood circulation is of great importance in studies of anticancer immunotherapy or drugs. However, the lack of a suitable imaging system hampers progress in this field. Methods: In this work, we built a dual-channel in vivo imaging flow cytometer to visualize the interactions of circulating tumor cells (CTCs) and dendritic cells (DCs) simultaneously in the bloodstream. Two artificial neural networks were trained to identify blood vessels and cells in the acquired images. Results and Conclusion: Using this technique, single CTCs and CTC clusters were readily distinguished by their morphology. Interactions of CTCs and DCs were identified, while their moving velocities were analyzed. The CTC-DC clusters moved at a slower velocity than that of single CTCs or DCs. This may provide new insights into tumor metastasis and blood rheology. Significance: This in vivo imaging flow cytometry system holds great potential for assessing the efficiency of targeting CTCs with anticancer immune cells or drugs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-γ, and tumor necrosis factor-α, 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 FROM AUTHOR]

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 FROM AUTHOR]