Label-free in-vivo classi-cation and tracking of red blood cells and platelets using Dynamic-YOLOv4 network

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-vivoflow cytometry,which holds promise for diverse in-vivo cell classification applications.

In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

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-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow 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.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Detection of cells by flow cytometry:Counting,imaging,and cell classification

The study of circulating cells in the blood stream is critical,as it covers many felds of biomed-icine,including immunology,cell biology,oncology,and reproductive medicine.In-viuo flowcytometry(IVFC)is a new tool to monitor and count cells in real time for long durations in theirnative biological environment.This review describes two main categories of IVFC,ie.,labeledand label-free IVFC.It focuses on label-free IVFC and introduces its technological developmentand related biological applications.Because cell recognition is the basis of flow cytometrycounting,this review also describes various methods for the classification of unlabeled cells,including the latest machine learning-based technologies.

Recent advances in Raman spectroscopy for skin diagnosis

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 specic 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 notications.Finally,we discuss the current limitations and future progress of Raman spectroscopy in the context of skin diagnosis.

Editorial Introduction to special issue on single cell analysis(Part Ⅱ)

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.The rst part of this special issue was published in March 2023,which includes three review and ve research articles.1 It is exciting for us to receive some positive responses from our peers who have continued to contributeve additional articles to form the single cell analysis(Part Ⅱ).We believe that these papers of this special issue will provide a better understanding of single cell analysis.

Introduction to special issue on single cell analysis

Increasing evidence has shown that cell populations are not homogeneous,but rather heterogeneous,even within very small cell populations.Bulk measurements based on the homogenized cell population do not account for the critical changes occurring in individual cells and are sometimes misleading.Cellular heterogeneity characteristics may be the key to address previously unsolved questions in disease development and progression.

Recent advances in fuorescence-based in vivo flow cytometry

The fuorescence-based in vivo flow cytometry(IVFC)is an emerging tool to monitor eirculating cells in vivo.As a noninvasive and real-timne 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(eg,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,difuse-light methods,hybrid multimodality methods and multispectral methods are also summarized.

Near infra-red light treatment of Alzheimer's disease

Alzheimers disease(AD)is a chronic neurodegenerative disease.The symptoms include memoryand spatial learning dificulties,language disorders,and loss of motivation,which get worse overtime,eventually ending in death.No ffective treatments are available for AD,currently.Currenttreatments only attenuate symptoms temporarily and are associated with severe side ffects.Nearinfra-red(NIR)light has been studied for a long time.We investigated the effect of NIR on ADusing a transgenic mouse model,which was obtained by co-injecting two vectors carrying ADmutations in amyloid precursor protein(APP)and presenilin-i(PSEN1)into C57BL/6J mice.The irradiation equipment consisted of an accommodating box and an LED array.The wave-length of NIR light emitted from LED was between 1040 nm and 1090 nm.The power densitydelivered at the level of the mice was approximately 15 mW/cm^(2),Firstly,we treated the micewith NIR for 40 days,Then,the irradiation was suspended for 28 days.Finally,another 15 daystreatment was brought to mice.We conducted Morris water maze and immunofluorescenceanalysis to evaluate the effects of treatment.Immunofuorescence analysis was based on mea-suring the quantity of plaques in mouse brain slices,Our results show that NIR light improvesmemory and spatial learning ability and reduces plaques moderately.NIR light represents apotential treatment for AD.

STUDYING THE ROLE OF MACROPHAGES IN CIRCULATING PROSTATE CANCER CELLS BY IN VIVO FLOW CYTOMETRY

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients.To metastasize,t he malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation.Macrophages appear to be directly involved in tumor progression and metastasis.However,the role of macrophages in affecting cancer metast asis has not been fully elucidated.Here,we have utilized an emerging technique,namely in vivo flow cytometry(IVFC)to study the depletion kinetics of circulating prostate cancer cells in mice and determine how depletion of macrophages by the liposome encapsulated clodronate affects the depletion kinetics.Our results show diferent depletion kinetics of PC-3 cells between the macrophagedeficient group and the control group.The number of circulating tumor cells(CTCs)in the macrophage-deficient group decreases in a slower manner compared to the control mice group.The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation.In addition,our imaging data suggest that macrophages might be able to arrest,phagocytose and digest PC-3 cells.Therefore,phagocy tosis may mainly contribute to the de-pletion kinetic diferences.The developed methods elaborated here would be useful to study the relationship between macr ophages and tumor metastasis in small animal cancer models.

Binary organic nanoparticles with enhanced reactive oxygen species generation capability for photodynamic therapy

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.

Algorithm to identify circulating tumor cell clusters using in vivo flow cytometer

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°ow 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°uorescent 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 identied 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.

Toluidine blue O and porphyrin-mediated photodynamic therapy on three main pathogenic bacteria of periodontitis using portable LED phototherapy device

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 gingitvalis(P.g.)and Aggregatibacter actinomycetercomitans(A.a-).The portable red light-ermitting 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 photosensit izers(porphyrin)under dfferent 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.

Automated retinal layer segmentation in optical coherence tomography images with intraretinal fluid

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.

ICAM-1 depletion in the center of immunological synapses is important for calcium releasing in T-cells

T-cell activation requires the formation of the immunological sy napse(IS)bet ween a T-cll and anantigen-presenting cell(AP C)to control the development of the adaptive immune response.How-ever,calcium release,an initial signal of T-cell activation,has been found to occur before IS for-mation.The mechanism for triggering the calcium signaling and relationship bet ween calciumrelease and IS format ion remains unclear.Herein,using live-cell imaging,we found that int ercellularadhesion molecule 1(ICAM-1),an essential mdlecule for IS formation,accumulated and then wasdepleted at the center of the synapse before complete IS formation.During the proces of ICAM1depletion,calcium was released.if ICAM-1 failed to be depleted from the center of the synapse,thesustained calcium signaling could not be induced.Moreover,depletion of ICAM-1 in ISs preferen-tially ccurred with the contact of antigen-specific T-cels and dendritic clls(DCs).Blocking thebinding ofICA M-1 and lymphocy te finction-associated antigen 1(LFA-1),ICAM-1 failed to depleteat the center of the synapse,and calcium release in T-clls decreased.In studying the mechanism ofhow the depletion ofiCA M1 could influence calcium release in T-clls,we found that the movementof ICAM-1 was associat ed with the localization of LFA-1 in the IS,which afected the localization ofcalcium microdomains,ORAIl and mitochondria in IS.Therefore,the depletion of ICAM-1 in the center of the synapse is an important factor for an initial sust ained calcium release in T-cells.

Cell counting for in vivo flow cytometry signals with baseline drift

In biomedical research fields,the in vio Aow 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).

PHOTOTHERAPY FOR MILD TO MODERATE ACNE VULGARIS WITH PORTABLE BLUE AND RED LED

In this paper,we studied portable blue and red light-emitting-diode(LED)light sources in phototherapy for mild to moderate acne vulgaris to evaluate the efficacy and tolerance of patients.Patients,randomly divided into blue and red groups,received either blue or red LED phototherapy twice a week for four weeks.After complete treatment,the number of lesions reduced by 71.4% in the blue group,in contrast to 19.5% in the red group.No obvious side effects were observed during and one month after the treatment,except for some mild dryness mentioned by several patients.

PD-L1 is a direct target of cancer-FOXP3 in pancreatic ductal adenocarcinoma(PDAC),and combined immunotherapy with antibodies against PD-L1 and CCL5 is effective in the treatment of PDAC

High expression of PD-L1 marks the poor prognosis of pancreatic ductal adenocarcinomas(PDAC).However,the regulatory mechanism of PD-L1 remains elusive.We recently reported that cancer Forkhead box protein 3(Cancer-FOXP3 or C-FOXP3)promoted immune evasion of PDAC by recruiting Treg cells into PDAC via upregulation of CCL5.In this study,we confirmed that PD-L1 was overexpressed in PDAC samples from two independent cohorts of patients with radical resection.Moreover,C-FOXP3 was colocalized and correlated with the expression of PD-L1 in tumor cells at the mRNA and protein levels,and this finding was confirmed by the The Cancer Genome Atlas(TCGA)database.Chromatin immunoprecipitation(ChIP)revealed that C-FOXP3 directly bound to the promoter region of PD-L1 in pancreatic cancer cells.Furthermore,overexpression of C-FOXP3 activated the luciferase reporter gene under the control of the PD-L1 promoter.However,mutation of the binding motif-a completely reversed the luciferase activity.In addition,C-FOXP3-induced upregulation of PD-L1 effectively inhibited the activity of CD8+T cells.Based on our recent finding that the CCL-5 antibody achieved a better response to PDAC models with high C-FOXP3 levels,we further demonstrated that the PD-L1 antibody strengthened the antitumor effect of CCL-5 blockade in xenograft and orthotopic mouse models with high C-FOXP3 levels.In conclusion,C-FOXP3 directly activates PD-L1 and represents a core transcription factor that mediates the immune escape of PDAC.Combined blockade of PD-L1 and CCL-5 may provide an effective therapy for patients with PDAC that have high C-FOXP3 levels.

Microglia modulation with 1070-nm light attenuates Aβ burden and cognitive impairment in Alzheimer’s disease mouse model

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 cerebralβ-amyloid(Aβ)burden,the hallmark of AD.The glial cells,including microglia and astrocytes,play important roles in Aβclearance.Our results show that 1070-nm light pulsed at 10 Hz triggers microglia rather than astrocyte responses in AD mice.The 1070-nm lightinduced microglia responses with alteration in morphology and increased colocalization with Aβare sufficient to reduce Aβload in AD mice.Moreover,1070-nm light pulsed at 10 Hz can reduce perivascular microglia and promote angiogenesis to further enhance Aβclearance.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.

Early changes to the extracellular space in the hippocampus under simulated microgravity conditions

The smooth transportation of substances through the brain extracellular space(ECS)is crucial to maintaining brain function;however,the way this occurs under simulated microgravity remains unclear.In this study,tracer-based magnetic resonance imaging(MRI)and DECS-mapping techniques were used to image the drainage of brain interstitial fluid(ISF)from the ECS of the hippocampus in a tail-suspended hindlimb-unloading rat model at day 3(HU-3)and 7(HU-7).The results indicated that drainage of the ISF was accelerated in the HU-3 group but slowed markedly in the HU-7 group.The tortuosity of the ECS decreased in the HU-3 group but increased in the HU-7 group,while the volume fraction of the ECS increased in both groups.The diffusion rate within the ECS increased in the HU-3 group and decreased in the HU-7 group.The alterations to ISF drainage and diffusion in the ECS were recoverable in the HU-3 group,but neither parameter was restored in the HU-7 group.Our findings suggest that early changes to the hippocampal ECS and ISF drainage under simulated microgravity can be detected by tracer-based MRI,providing a new perspective for studying microgravity-induced nano-scale structure abnormities and developing neuroprotective approaches involving the brain ECS.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells

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.Fluoresce nee in vivo flow cytometry(IVFC)is a powerful optical technique that is able to detect fluoresce nt 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.