Initial refinement of data from video‐based single‐cell tracking

Background: Video recording of cells offers a straightforward way to gainvaluable information from their response to treatments. An indispensable stepin obtaining such information involves tracking individual cells from therecorded data. A subsequent step is reducing such data to represent essentialbiological information. This can help to compare various single‐cell trackingdata yielding a novel source of information. The vast array of potential datasources highlights the significance of methodologies prioritizing simplicity,robustness, transparency, affordability, sensor independence, and freedomfrom reliance on specific software or online services.Methods: The provided data presents single‐cell tracking of clonal (A549)cells as they grow in two‐dimensional (2D) monolayers over 94 hours,spanning several cell cycles. The cells are exposed to three differentconcentrations of yessotoxin (YTX). The data treatments showcase theparametrization of population growth curves, as well as other statisticaldescriptions. These include the temporal development of cell speed in familytrees with and without cell death, correlations between sister cells, single‐cellaverage displacements, and the study of clustering tendencies.Results: Various statistics obtained from single‐cell tracking reveal patternssuitable for data compression and parametrization. These statistics encompassessential aspects such as cell division, movements, and mutual informationbetween sister cells.Conclusion: This work presents practical examples that highlight theabundant potential information within large sets of single‐cell tracking data.Data reduction is crucial in the process of acquiring such information whichcan be relevant for phenotypic drug discovery and therapeutics, extendingbeyond standardized procedures. Conducting meaningful big data analysistypically necessitates a substantial amount of data, which can stem fromstandalone case studies as an initial foundation.

Computerized cell tracking:Current methods,tools and challenges

In developmental biology,knowledge of cell structure and their(morpho)dynamic behavior,leads to a comprehensive understanding of their conducts and the mechanisms in which they participate.This knowledge is a decisive factor in biological research and also in all drug development steps,medicinal or preventive therapies.Experimental cell analysis is hard,expensive,and time-consuming.To overcome these difficulties,in recent years,several computational object tracking methods,software system and packages have been developed in cell sciences that bring together different disciplines and branches of technologies.Object tracking is the process of locating and monitoring specific object and its behavior in sequential images.In this paper,a comprehensive review on object tracking stages and computational methods that are utilized in terms of cell tracking has been organized.Besides,the available software packages and toolkits,challenges,and their solution in time lapse microscopy images in this scope were reviewed.The aim of describing computational cell tracking methods and tools is that biologist and cell scientists might take advantage of these computational techniques to find another method to gain complementary information for their question of interest.

Dynamic tracking of stem cells in an acute liver failure model

AIM： To investigate a dual labeling technique, which would enable real-time monitoring of transplanted em- bryonic stem cell （ESC） kinetics, as well as long-term tracking. METHODS： Liver damage was induced in C57/BL6 male mice （n = 40） by acetaminophen （APAP） 300 mg/kg administered intraperitoneally. Green fluores- cence protein （GFP） positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbo- cyanine iodide （DiR） immediately before transplantationinto the spleen. Each of the animals in the cell therapy group （n = 20） received 5 x 106 ESCs 4 h following treatment with APAP. The control group （n = 20） re- ceived the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS lumina-2 at 30 rain post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohisto- chemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine amino- transferase （ALT） was measured as an indication of liver damage.RESULTS： DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradu- ally moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imag- ing, and confirmed that the highest photon emission was in the liver （P 〈 0.0001）. At 2 wk, the DiRsignal was no longer detectable in vivo; however, immuno- histochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP ＋ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP ＋ve cells incorporated within the sinu- soidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP ＋re cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly （P = 0.016） lower and al- bumin （P = 0.009） was significantly higher when com- pared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups. CONCLUSION： Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribu- tion, survival and engraftment of transplanted cells, and could be used for cell therapy models.

Nanotechnology-assisted adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury： In vivo cell tracking, optimized injection dosage, and functional evaluation

Stem cell therapy is a potentially promising option for erectile dysfunction; however, its risk of tumorigenicity is a clinical hurdle and the risk is positively related to the number of injected cells. Our previous study showed that nanotechnology improved adipose-derived stem cell （ADSC） therapy for erectile dysfunction of cavernous nerve injury （CNI） by attracting cells in the corpus cavernosum. These results indicated the possibility of using a reduced dosage of ADSCs for intracavernous injection. In this exploratory study, we used lower dosage （2 × 105 cells） of ADSCs for intracavernous injection （ICI） and the nanotechnology approach. Intracavernous pressure and mean arterial pressure were measured at day 28 to assess erectile function. The low-dose ADSC therapy group showed favorable treatment effects, and nanotechnology further improved these effects. In vivo imaging of ICI cells revealed that the fluorescein signals of NanoShuttle-bound ADSCs （NanoADSCs） were much stronger than those of ADSCs at days 0, 1, and 3. Both immunofluorescence and Western blot analysis showed a significant increase in smooth muscle, endothelium, and nerve tissue in the ADSC group compared to that in the CNI group; further improvement was achieved with assisted nanotechnology. These findings demonstrate that nanotechnology can be used to further improve the effect of small dosage of ADSCs to improve erectile function. Abundant NanoADSCs remain in the corpus cavernosum in vivo for at least 3 days. The mechanism of erectile function improvement may be related to the regeneration of the smooth muscle, endothelium, and nerve tissues.

Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

Tracking of Labelled Stem Cells Using Molecular MR Imaging in a Mouse Burn Model <i>in Vivo</i>as an Approach to Regenerative Medicine

Therapies based on stem cell transplants offer significant potential in the field of regenerative medicine. Monitoring the fate of the transplanted stem cells in a timely manner is considered one of the main limitations for long-standing success of stem cell transplants. Imaging methods that visualize and track stem cells in vivo non-invasively in real time are helpful towards the development of successful cell transplantation techniques. Novel molecular imaging methods which are non-invasive particularly such as MRI have been of great recent interest. Hence, mouse models which are of clinical relevance have been studied by injecting contrast agents used for labelling cells such as super-paramagnetic iron-oxide (SPIO) nanoparticles for cellular imaging. The MR techniques which can be used to generate positive contrast images have been of much relevance recently for tracking of the labelled cells. Particularly when the off-resonance region in the vicinity of the labeled cells is selectively excited while suppressing the signals from the non-labeled regions by the method of spectral dephasing. Thus, tracking of magnetically labelled cells employing positive contrast in vivo MR imaging methods in a burn mouse model in a non-invasive way has been the scope of this study. The consequences have direct implications for monitoring labeled stem cells at some stage in wound healing. We suggest that our approach can be used in clinical trials in molecular and regenerative medicine.

Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models

BACKGROUND Chronic liver diseases(CLD)are the major public health burden due to the continuous increasing rate of global morbidity and mortality.The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option.However,identifying the fate of transplanted cells in vivo represents a crucial obstacle.AIM To evaluate the potential applicability of DiD dye as a cell labeling agent for longterm,and non-invasive in vivo tracking of transplanted cells in the liver.METHODS Magnetically sorted,epithelial cell adhesion molecule positive(1×106 cells/mL)fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency(SCID)mice.Near-infrared(NIR)imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d.The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.RESULTS NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7,15,30,45,60,and 80 post-transplantation.Furthermore,positive staining of cytokeratin,c-Met,and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells.Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase,glutamate-pyruvate transaminase,and bilirubin.The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.CONCLUSION DiD-labeling is promising for long-term and non-invasive in vivo cell tracking,and understanding the regenerative mechanisms incurred by the transplanted cells.

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.

MRI/PAI Dual-modal Imaging-guided Precise Tracking of Bone Marrow-derived Mesenchymal Stem Cells Labeled with Nanoparticles for Treating Liver Cirrhosis

Background and Aims:Stem cell transplantation is a potential treatment option for liver cirrhosis(LC).Accurately and noninvasively monitoring the distribution,migration,and prognosis of transplanted stem cells using imaging methods is important for in-depth study of the treatment mechanisms.Our study aimed to develop Au-Fe3O4 silica nanoparticles(NPs)as tracking nanoplatforms for dualmodal stem cell imaging.Methods:Au-Fe3O4 silica NPs were synthesized by seed-mediated growth method and co-precipitation.The efficiency and cytotoxicity of the NPslabeled bone marrow-derived mesenchymal stem cells(BMMSCs)were evaluated by Cell Counting Kit-8 assays,ICPMS,phenotypic characterization,and histological staining.The biodistribution of labeled BM-MSCs injected through different routes(the hepatic artery or tail vein)into rats with LC was detected by magnetic resonance imaging(MRI),photoacoustic imaging(PAI),and Prussian blue staining.Results:Synthesized Au-Fe3O4 silica NPs consisted of a core(star-shaped Au NPs)and an outside silica layer doped with Fe3O4 NPs.After 24 h coincubation with 2.0 OD concentration of NPs,the viability of BM-MSCs was 77.91%±5.86%and the uptake of Au and Fe were(22.65±1.82)µg/mL and(234.03±11.47)µg/mL,respectively.The surface markers of labeled BM-MSCs unchanged significantly.Labeled BMMSCs have osteogenic and adipogenic differentiation potential.Post injection in vivo,rat livers were hypointense on MRI and hyperintense on PAI.Prussian blue staining showed that more labeled BM-MSCs accumulated in the liver of the hepatic artery group.The severity of LC of the rats in the hepatic artery group was significantly alleviated.Conclusions:Au-Fe3O4 silica NPs were suitable MRI/PAI dual-modal imaging nanoplatforms for stem cell tracking in regenerative medicine. Transhepatic arterial infusion of BMMSCs was the optimal route for the treatment of LC.

Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina

Background Endothelial progenitor cells （EPCs） transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester （CFSE） in laser-injured mouse retina. Methods EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 μmol/L CFSE at 37℃ was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.Results EPCs labeled with 5 μmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks, The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.Conclusion EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.

Adaptive Cell Segmentation and Tracking for Volumetric Confocal Microscopy Images of a Developing Plant Meristem

Automated segmentation and tracking of cells in actively developing tissues can provide high-throughput and quantitative spatiotemporal measurements of a range of cell behaviors; cell expansion and cell-division kinetics leading to a better understanding of the underlying dynamics of morphogenesis. Here, we have studied the problem of constructing cell lineages in time-lapse volumetric image stacks obtained using Confocal Laser Scanning Microscopy （CLSM）. The novel contribution of the work lies in its ability to segment and track cells in densely packed tissue, the shoot apical meristem （SAM）, through the use of a close-loop, adaptive segmentation, and tracking approach. The tracking output acts as an indicator of the quality of segmentation and, in turn, the segmentation can be improved to obtain better tracking results. We construct an optimization function that minimizes the segmentation error, which is, in turn, estimated from the tracking results. This adaptive approach significantly improves both tracking and segmentation when compared to an open loop framework in which segmentation and tracking modules operate separately.

Regulation of cell locomotion by nanosecond-laser-induced hydroxyapatite patterning

Hydroxyapatite,an essential mineral in human bones composed mainly of calcium and phosphorus,is widely used to coat bone graft and implant surfaces for enhanced biocompatibility and bone formation.For a strong implant-bone bond,the bone-forming cells must not only adhere to the implant surface but also move to the surface requiring bone formation.However,strong adhesion tends to inhibit cell migration on the surface of hydroxyapatite.Herein,a cell migration highway pattern that can promote cell migration was prepared using a nanosecond laser on hydroxyapatite coating.The developed surface promoted bone-forming cell movement compared with the unpatterned hydroxyapatite surface,and the cell adhesion and movement speed could be controlled by adjusting the pattern width.Live-cell microscopy,cell tracking,and serum protein analysis revealed the fundamental principle of this phenomenon.These findings are applicable to hydroxyapatite-coated biomaterials and can be implemented easily by laser patterning without complicated processes.The cell migration highway can promote and control cell movement while maintaining the existing advantages of hydroxyapatite coatings.Furthermore,it can be applied to the surface treatment of not only implant materials directly bonded to bone but also various implanted biomaterials implanted that require cell movement control.

Fucoidan-functionalized activated platelet-hitchhiking micelles simultaneously track tumor cells and remodel the immunosuppressive microenvironment for efficient metastatic cancer treatment

Tumor metastasis is responsible for most mortality in cancer patients, and remains a challenge in clinical cancer treatment. Platelets can be recruited and activated by tumor cells, then adhere to circulating tumor cells(CTCs) and assist tumor cells extravasate in distant organs. Therefore, nanoparticles specially hitchhiking on activated platelets are considered to have excellent targeting ability for primary tumor, CTCs and metastasis in distant organs. However, the activated tumor-homing platelets will release transforming growth factor-β(TGF-β), which promotes tumor metastasis and forms immunosuppressive microenvironment. Therefore, a multitalent strategy is needed to balance the accurate tumor tracking and alleviate the immunosuppressive signals. In this study, a fucoidan-functionalized micelle(FD/DOX) was constructed, which could efficiently adhere to activated platelets through P-selectin.Compared with the micelle without P-selectin targeting effect, FD/DOX had increased distribution in both tumor tissue and metastasis niche, and exhibited excellent anti-tumor and anti-metastasis efficacy on 4 T1 spontaneous metastasis model. In addition, due to the contribution of fucoidan, FD/DOXtreatment was confirmed to inhibit the expression of TGF-β, thereby stimulating anti-tumor immune response and reversing the immunosuppressive microenvironment. The fucoidan-functionalized activated platelets-hitchhiking micelle was promising for the metastatic cancer treatment.

Highly-Sensitive Multiplexed in vivo Imaging Using PEGylated Upconversion Nanoparticles

Lanthanide-based upconversion nanoparticles(UCNPs)have been widely explored in various fields,including optical imaging,in recent years.Although earlier work has shown that UCNPs with different lanthanide(Ln3+)dopants exhibit various colors,multicolor-especially in vivo multiplexed biomedical imaging-using UCNPs has rarely been reported.In this work,we synthesize a series of UCNPs with different emission colors and functionalize them with an amphiphilic polymer to confer water solubility.Multicolor in vivo upconversion luminescence(UCL)imaging is demonstrated by imaging subcutaneously injected UCNPs and applied in multiplexed in vivo lymph node mapping.We also use UCNPs for multicolor cancer cell labeling and realize in vivo cell tracking by UCL imaging.Moreover,for the first time we compare the in vivo imaging sensitivity of quantum dot(QD)-based fluorescence imaging and UCNP-based UCL imaging side by side,and find the in vivo detection limit of UCNPs to be at least one order of magnitude lower than that of QDs in our current non-optimized imaging system.Our data suggest that,by virtue of their unique optical properties,UCNPs have great potential for use in highly-sensitive multiplexed biomedical imaging.