Cell Imaging Research Articles

Abstract A035: 3D High-Density Collagen I Improves the Modeling of Aggressive Pancreatic Cancer

Abstract Background: Fibrillar collagen type I constitutes the majority of the extracellular matrix in pancreatic ductal adenocarcinoma (PDAC), yet most in vivo murine models involve the use of basement membrane extracts, which instead contain collagen type IV, as matrices to embed PDAC cells. Type I collagen in the PDAC tumor microenvironment has been associated with worse prognosis. Our hypothesis is that PDAC cells grown in 3D high-density collagen I (HDC) gels behave more aggressively compared to the same cells grown using conventional basement membrane matrix (BMM). Methods: The KPC1199 2D cell line was previously generated from a tumor-bearing male KPC mouse. KPC1199 cells were cultured in vitro in HDC or BMM for one week. Orthotopic tumors were established by embedding KPC1199 cells in HDC (N=10) or BMM (N=10) in the pancreatic tail of syngeneic C57BL/6J mice via laparotomy. Half of the tumors were collected five weeks after tumor implantation, and the other half utilized for a survival study. Collagen was imaged by second harmonic generation (SHG) microscopy. Results: In vitro imaging of single KPC1199 cells grown in HDC revealed an aggressive phenotype with clonal network-like structures compared to a spheroidal, non-aggressive phenotype for cells grown in BMM over the course of seven days. SHG imaging confirmed consistent formation and organization of collagen in HDC tumors but minimal to faint collagen formation in BMM tumors. Gross examination of HDC mice showed 60% with visible liver metastasis compared with none in the BMM mice. In the survival study, the entire HDC group died before the BMM group (p-value < 0.01). Conclusions: Our pilot study is the first, to our knowledge, to successfully generate orthotopic PDAC tumors using 3D high-density collagen I in a mouse model. KPC1199 cells embedded in HDC showed an aggressive phenotype in vitro, and resultant tumors demonstrated more robust collagen organization compared to the same cells grown in the BMM substrate. This aggressive phenotype translated to visible liver metastasis within five weeks in the HDC mice and prior to death from primary tumor burden. This is not typical for most KPC-derived cell lines but is typical for human PDAC patients. Our preliminary findings support the use of HDC as an alternative substrate for better modeling of this aggressive cancer. Citation Format: Kim Nguyen-Ta, Sural Ranamukhaarachchi, Jeffrey Turner, Utsav Joshi, Himangshu Sonowal, Jorge de la Torre Medina, Herve Tiriac, Stephanie Fraley, Rebekah White. 3D High-Density Collagen I Improves the Modeling of Aggressive Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A035.

Abstract B076: Morphological diversity predicts functional traits in pancreatic cancer

Abstract Cell morphologies represent a phenotypic integration of intrinsic cellular processes, changes in cell state, and interactions with neighboring cells. While analyses of large cell line collections such as the Cancer Dependency Map (DepMap) have greatly enhanced discovery of novel molecular biomarkers and therapeutic targets, morphological analysis of these models has not yet been performed. Hence, there is a rich opportunity to discover novel regulators and functional properties of cell morphologies by linking image-based profiling to -omics based assays. Here, we studied the transcriptional and functional properties of 16 human pancreatic cancer cell lines, associations with distinct cell morphologies, and treatment-induced remodeling. At baseline, we observed extensive morphological heterogeneity across and within cell lines, as well as differences in multicellular organization. We categorized cell line morphologies into epithelial, mesenchymal, and spheroid, and organizational patterns into tightly aggregated, multilayered, and dispersed. We performed differential expression analysis across morphological subtypes utilizing DepMap transcriptomics data and queried functional correlates including CRISPR dependency, drug sensitivity, and proclivity for metastasis. For example, we discovered that the mesenchymal morphology and multilayered organizational pattern [WH3] were associated with metastatic potential, while expressing reduced levels of tight junction and cell adhesion genes. We then explored treatment associated morphological changes and their potential as cost-effective biomarkers for cell-intrinsic resistance to chemotherapy (e.g., 5-FU and gemcitabine) and KRAS inhibitors (e.g., MRTX1133 and RMC- 6236). Cells were treated continuously and monitored with live cell imaging (Incucyte SX5) followed by Cell Painting at the three-day endpoint. We observed conserved morphological responses to therapy across cell lines including a shift towards spindle-like cell shapes with neurite-like projections in chemotherapy-treated conditions, and a cell bloating phenomena characterized by an increase in cell area in KRAS inhibitor treated conditions. To directly link transcriptional state to morphology, we performed 1000-plex spatial molecular imaging (Nanostring CosMx) at subcellular resolution to pools of cell lines, demonstrating that morphological diversity within cell lines corresponded to distinct transcriptional states. In conclusion, this study highlights the potential of harnessing cell morphological information in a rapid, cost-effective phenotyping assay to aid precision oncology efforts leveraging patient- derived in vitro models. Citation Format: Dennis Gong, William L Hwang. Morphological diversity predicts functional traits in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B076.

Unveiling Hierarchical Self-Assembly of Triazolylferrocenyl Dendrimers: Producing Non-Traditional Intrinsically Green Fluorescent Vesosomes for Nanotheranostics.

Dendrimers and supramolecular chemistry continue to fascinate researchers due to the endless unrevealed potential of their combination. This study investigates the self-assembly process of a series of hydrophobic triazolylferrocenyl dendrimers in aqueous medium. Deep investigation through NMR spectroscopy, absorption UV-vis spectroscopy along with theoretical simulations demonstrates that the ferrocenyl moieties interact intramolecularly and intermolecularly driving the self-assembly process. Data obtained by DLS, NTA, SEM, TEM, and EF-TEM demonstrate that these dendrimers, in water, spontaneously self-assemble through a hierarchical process. The dendrimers first self-assemble into uniform nanovesicles, which in turn self-assemble into larger vesosomes. The resulting vesosomes emit green non-traditional intrinsic fluorescence, which is a property that emerged from the self-assembled architectures. The vesosomes are efficiently uptaken by cancer cells and induce significant cytotoxic activity against the cancer cell line MCF-7, up to the submicromolar concentration. Positive dendritic effects are identified in the fluorescence intensity and in the cytotoxic activity of the vesosomes, which follow the trend G0-9Fc < G1-27Fc < G2-81Fc. This work showcases the remarkable potential of combining the two dynamic fields of dendrimers and supramolecular chemistry, which resulted in green fluorescent vesosomes capable of performing the dual role of cell imaging and killing, with potential applications in nanotheranostics.

Classification of cervical cells from the Pap smear image using the RES_DCGAN data augmentation and ResNet50V2 with self-attention architecture

AbstractCervical cancer is a type of cancer in which abnormal cell growth occurs on the surface lining of the cervix. In this study, we propose a novel residual deep convolutional generative adversarial network (RES_DCGAN) for data augmentation and ResNet50V2 self-attention method to classify cervical cells, to improve the generalizability and performance of the model. The proposed method involves adding residual blocks in the generator of the DCGAN to enhance data flow and generate higher-quality images. Subsequently, a self-attention mechanism is incorporated at the top of the pre-trained models to allow the model to focus more on significant features of the input data. To evaluate our approach, we utilized the Pomeranian and SIPaKMeD cervical cell imaging datasets. The results demonstrate superior performance, achieving an accuracy of 98% with Xception and 96.4% with ResNet50V2 on the Pomeranian dataset. Additionally, DenseNet121 with self-attention achieved accuracies of 92% and 95% in multiclass and binary classification, respectively, using the SIPaKMeD dataset. In conclusion, our RES_DCGAN-based data augmentation and pre-trained with self-attention model yields a promising result in the classification of cervical cancer cells.

A Water‐Soluble Fluorescent Probe Derived from Pyridine‐2,6‐Dicarboxylic Acid: Synthesis, Interaction with Ions, and Two‐Photon Microscopy

AbstractTwo‐photon excited fluorescence is a process in which the excited fluorophore relaxes and emits fluorescence after two‐photon absorption. Combined with appropriate two‐photon fluorescent probes, two‐photon microscopy enables observation deep inside tissue without damage. Former reports showed that the two‐photon absorption cross sections of donor–acceptor (D–A) dipoles, as well as D‐π‐D and D–A–D quadrupoles can be strengthened by using strong D–A groups. In this study, a novel water‐soluble fluorescent probe with D‐π‐A structure containing pyridine‐2,6‐dicarboxylic acid unit was synthesized. The maximum excitation wavelength and emission wavelength of the probe in aqueous solution were 328 nm and 471 nm, respectively. The fluorescence emitted by the probe can be quenched by various metal cations. The pyridyl N and carboxyl O in the probe formed chelates with Cu2+, which resulted in a fluorescence quenching response, and showed a linear relationship between fluorescent intensity and Cu2+ concentration with wide linear range and low limit of detection. CCK‐8 tests showed the probe had no obvious toxicity to 4T1 cells in the range of 0.4–50 μmol L−1, and the cell survival rate was above 86 %. The probe had a large two‐photon absorption cross section ranging from 680 nm to 820 nm. The two‐photon absorption cross section reached 100 GM at 760 nm of excitation wavelength. By means of microscopic imaging of 4T1 cells stained with the probe, one‐photon fluorescence represented blue emission, while two‐photon fluorescence represented bright green emission. The probe was proved to have good cell permeability, and preferred to target nucleus, showing application potential in two‐photon fluorescence imaging technology.

Leukocyte classification using relative-relationship-guided contrastive learning

Hematologic diseases and blood disorders can be studied through microscopic examination of blood smear images or chemical assays. Many researchers are focused on utilizing deep learning (DL) to identify, quantify, and classify various types of blood cells, which is crucial for addressing theoretical and practical challenges in disease diagnosis and treatment planning. However, there is a significant deficiency in annotations for leukocyte classification, limiting the effectiveness of current DL methods. Contrastive learning (CL) facilitates the extraction of prior knowledge from unlabeled data, reducing the dependency on manual annotations. While CL demonstrates remarkable achievements in classifying natural images, the direct application to leukocyte classification presents certain defects. We observe that standard data augmentation techniques in CL exhibit varying sensitivity to different categories of white blood cell images. Employing a uniform augmentation strategy may lead the network into an optimization trap, acquiring inadequate representations from erroneous sample relationships. To address this issue, we propose a Relative-Relationship-Guided Contrastive Learning Representation (ReCLR) framework for the leukocyte classification. ReCLR mines positive and negative samples based on relative distance knowledge. Specifically, we provide adversarial guidance for the positive sample, restricting the distance between the positive sample and the original sample less than but as close as possible to the distance between the original sample and the farthest negative sample. Then, we utilize the entropy constraint to regulate the relative distance relationship between the negative and original samples. Finally, the guided positive and negative samples are employed for contrastive learning in leukocyte classification. The extensive experiments, including linear evaluation, domain transfer, and fine-tuning, demonstrate the effectiveness of the proposed method. Our ReCLR achieves accuracies of 92.07%, 65.36%, and 92.49% on three real-world leukocyte datasets, respectively, outperforming several state-of-the-art methods. The source code is released at https://github.com/AlchemyEmperor/ReCLR.

Thiophene appended schiff base probe for selective ‘turn-on’ response for copper (ii) ion and their applications in live cell imaging Thiophene appended schiff base probe for selective ‘turn-on’ response for copper (ii) ion and their applications in live cell imaging

In this research, we have synthesized a thiophene Schiff base from 5-phenyl thiophene-2-carboxaldehyde and 2-thiophene carboxylic acid hydrazide and characterized by using FT-IR NMR, HRMS and SC-XRD analysis techniques. The probe exhibited a selective turn-on fluorescence response for Cu2+ over 20 other common metal ions in a CH3CN (9:1, v/v) solution. The Job’s plot represents a 1:1 binding complexation mode and further DFT study was carried out to aid in understanding the geometric structure and interaction of the ligand and Cu2+. The detection limit (LOD) and limit of quantification (LOQ) were found to be 20 nM and 69 nM respectively. Furthermore, the probe’s sensing ability was tested in HeLa cells and their fluorescence imaging also supported the effective turn-on response towards Cu2+ ion.

Differential interference with actin-binding protein function by acute Cytochalasin B.

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa , and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding β-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

Streptococcus suis Induces Macrophage M1 Polarization and Pyroptosis.

Streptococcus suis is an important bacterial pathogen that affects the global pig industry. The immunosuppressive nature of S. suis infection is recognized, and our previous research has confirmed thymus atrophy with a large number of necrotic cells. In this current work, we aimed to uncover the role of pyroptosis in cellular necrosis in thymic cells of S. suis-infected mice. Confocal microscopy revealed that S. suis activated the M1 phenotype and primed pyroptosis in the macrophages of atrophied thymus. Live cell imaging further confirmed that S. suis could induce porcine alveolar macrophage (PAM) pyroptosis in vitro, displaying cell swelling and forming large bubbles on the plasma membrane. Meanwhile, the levels of p-p38, p-extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) were increased, which indicated the mitogen-activated protein kinase (MAPK) and AKT pathways were also involved in the inflammation of S. suis-infected PAMs. Furthermore, RT-PCR revealed significant mRNA expression of pro-inflammatory mediators, including interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α and chemokine CXCL8. The data indicated that the inflammation induced by S. suis was in parallel with pro-inflammatory activities of M1 macrophages, pyroptosis and MAPK and AKT pathways. Pyroptosis contributes to necrotic cells and thymocyte reduction in the atrophied thymus of mice.

An intelligent NIR fluorescent dye with dual response to pH and viscosity for investigating the interactions between LDs and mitochondria

The interaction between lipid droplets and mitochondria plays a pivotal role in biological processes including cellular stress, metabolic homeostasis, cellular autophagy and apoptosis. Deciphering the complex interplay between lipid droplets and mitochondria is essential for gaining insights into the fundamental workings of the cell and can have broad implications for the development of therapeutic strategies for various diseases, including metabolic disorders, neurodegenerative diseases, and cancer. In this study, we develop a pH and viscosity-responsive near-infrared (NIR) fluorescent probe PTOH to investigate the interaction between lipid droplets and mitochondria. This probe demonstrates a significant enhancement in fluorescence intensity at 470 nm when the pH increases, while under acidic conditions, its fluorescence intensity at 730 nm intensifies by a factor of 35 with rising system viscosity. Cell imaging experiments revealed that PTOH can effectively discriminate between normal and cancerous cells, as well as detect intracellular pH and viscosity alterations induced by drugs. Additionally, PTOH is utilized to visualize the interaction between lipid droplets and mitochondria and to differentiate between cellular autophagy and apoptosis phenomena, providing a valuable tool for elucidating the mechanisms underlying lipid droplet-mitochondria interactions and their associated diseases.

A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs.

A naphthalimide-based bifunctional fluorescent probe for detecting CN− and alkaline pH and its application in food samples and living cells imaging

Cyanide (CN−), as one of the most deadly toxic substances, has further promoted the development of fluorescent probes for identifying it, but the fluorescent probes capable of simultaneously identifying cyanide and alkaline pH have always been a problem. Herein, we have synthesized a novel bifunctional fluorescent probe NCCQ for detecting CN− and alkaline pH. After the reaction of probe NCCQ with CN−, the fluorescence quenching phenomenon occurred obviously, and the quenching efficiency reached 82.7 %. NCCQ is sensitive to CN− (LOD = 44.5 nm), has good selectivity and excellent anti-interference, and can realize rapid response to CN− (50 s). Due to the uncertainty of CN− addition, we further determined the product structure after CN− addition through theoretical calculation. In alkaline pH environment, the fluorescence intensity was further enhanced and red shifted by 14 nm, and the color changed from cyan to bright green. Through mass spectrometry titration experiment, 1HNMR titration experiment and theoretical calculation, the mechanism of detecting CN− and alkaline pH with NCCQ is fully and multi-dimensionally proved. To our surprise, NCCQ can not only be used to detect CN− in real water samples and food samples, but also be used to make bifunctional fluorescent test strips with good results. Due to its good biocompatibility, NCCQ has been successfully applied to the biological imaging of exogenous CN− in Hela, which further proves its great application potential in the fields of environment, food and biological analysis.

Cascade–Assembly–Confined Multilevel Supramolecular Assemblies for Multicolor Luminescence Information Storage and Cells Imaging Based on Slipped Pseudo[5]rotaxane

Multilevel green fluorescent supramolecular assemblies that derived from macrocyclic confinement–activated yellow fluorescent pseudo[5]rotaxane and subsequently assembled with anionic cyclodextrin derivative sulfobutylether–β–cyclodextrin (SBE–β–CD) were reported. Possessing four cationic coumarin arms, the coumarin–modified dibenzo–24–crown–8 guest (G) could be encapsulated by cucurbit[7]uril (CB[7]) at a 4:1 host–guest stoichiometry to form pseudo[5]rotaxane (G@CB[7]4) in the manner of dynamic slipping, which not only extended the fluorescence intensity of G by 53 times, but also increased the fluorescence lifetime by 5.8–fold from 0.65 to 3.76 ns. Subsequently, SBE–β–CD was co–assembled with G@CB[7]4 to control the topological morphology from small nanoparticles to larger ones, further enhancing fluorescence by 9.9 times along with a blue shift from 530 nm to 515 nm through cascade confinement. Compared with the direct assembly of G and SBE–β–CD through electrostatic interactions, only slight fluorescence increase was shown. After doping the NIR dye sulforhodamine 101 (SR101) acceptor, the G@CB[7]4@SBE–β–CD/SR101 exhibited high energy transfer efficiency to 79%, accompanied by the color changed from green to red under 365 nm irradiation. Moreover, the multicolor tunable emission could be achieved by adding dibenzylammonium chloride to the solution of G@CB[7]4, G@CB[7]4@SBE–β–CD and G@CB[7]4@SBE–β–CD/SR101. Ultimately, the final assemblies were used in multicolor luminescence information storage and cells imaging.

Fluorescence and Circular Dichroism Dual-Mode Probe for Chiral Recognition of Tyrosine and Its Applications in Bioimaging.

Chiral amino acids (AAs) are essential in metabolism and understanding physiological processes, and they could be used as biomarkers for the diagnosis of different diseases. In this study, chiral Cdots@Van were prepared by postmodifying an achiral Cdots core with vancomycin for recognizing and determining the enantiomeric excess (ee) of tyrosine (Tyr) enantiomers. The fluorescence response of Cdots@Van is based on an "on-off" strategy, with different quenching percentages for d- and l-tyrosine. Interestingly, the circular dichroism (CD) spectrum of Cdots@Van responded to only one form of Tyr enantiomer, specifically d-Tyr, and remained nearly unchanged upon the addition of l-Tyr. Quantum mechanical (QM) calculations were in excellent agreement with the experimental results, confirming the stronger binding affinity of Cdots@Van for d-Tyr compared to l-Tyr. We further investigated the chiral recognition ability of the interconnected vancomycin particles, which was synthesized using the EDC/NHS coupling reaction between vancomycin molecules without a Cdots core. Surprisingly, unlike free vancomycin molecules, interconnected vancomycin displayed an enantiomeric recognition ability by CD spectroscopy, similar to what was observed for Cdots@Van. Crucially, this chiral probe has been successfully utilized for cell imaging applications.

Design and synthesis of a TICT-based red-emissive fluorescent probe for the rapid and selective detection of HSA in human biofluids and live cell imaging.

Here, we report the design and synthesis of a D⋯π⋯A-based fluorescent probe, (E)-4-(4-(dibutylamine)-2-hydroxystyryl)-1-methylquinolin-1-ium (DHMQ), which is nonfluorescent in ∼100% PBS buffer medium due to a twisted intra molecular charge transfer (TICT) phenomenon and it becomes highly fluorescent (∼149 fold) in the presence of human serum albumin (HSA), owing to the restriction of its intramolecular free rotation inside the hydrophobic binding cavity of HSA. The site-selective fluorescence displacement assay and molecular docking studies clearly reveal that DHMQ selectively binds at subdomain IB of HSA. The 3σ/slope method was adopted to determine the limit of detection (LOD) value, which was as low as 2.39 nM in ∼100% PBS medium, indicating its high sensitivity towards HSA. The low dissociation constant value [Kd = (1.066 ± 0.017) μM] suggests a strong complexation between the DHMQ and HSA. Importantly, it has been demonstrated that DHMQ is capable of detecting HSA in real human serum and urine samples and was found to be suitable for live cell imaging of HSA.

Practical guidelines for cell segmentation models under optical aberrations in microscopy

Cell segmentation is essential in biomedical research for analyzing cellular morphology and behavior. Deep learning methods, particularly convolutional neural networks (CNNs), have revolutionized cell segmentation by extracting intricate features from images. However, the robustness of these methods under microscope optical aberrations remains a critical challenge. This study evaluates cell image segmentation models under optical aberrations from fluorescence and bright field microscopy. By simulating different types of aberrations, including astigmatism, coma, spherical aberration, trefoil, and mixed aberrations, we conduct a thorough evaluation of various cell instance segmentation models using the DynamicNuclearNet (DNN) and LIVECell datasets, representing fluorescence and bright field microscopy cell datasets, respectively. We train and test several segmentation models, including the Otsu threshold method and Mask R-CNN with different network heads (FPN, C3) and backbones (ResNet, VGG, Swin Transformer), under aberrated conditions. Additionally, we provide usage recommendations for the Cellpose 2.0 Toolbox on complex cell degradation images. The results indicate that the combination of FPN and SwinS demonstrates superior robustness in handling simple cell images affected by minor aberrations. In contrast, Cellpose 2.0 proves effective for complex cell images under similar conditions. Furthermore, we innovatively propose the Point Spread Function Image Label Classification Model (PLCM). This model can quickly and accurately identify aberration types and amplitudes from PSF images, assisting researchers without optical training. Through PLCM, researchers can better apply our proposed cell segmentation guidelines. This study aims to provide guidance for the effective utilization of cell segmentation models in the presence of minor optical aberrations and pave the way for future research directions.

Integrated multiscale analysis reveals complex gender-specific changes in lymphocytes of smokers

Environmental stressors induce specific physiological responses that can be measured in the blood, notably by morphological changes in lymphocytes. Tobacco being the best-known stress in terms of its impact on health, we studied the physiological properties of peripheral blood lymphocytes in a population of 33 healthy non-smokers and smokers. Proteasome amount, mitochondria energy levels, changes in membrane properties and cell and nuclear size were analyzed to obtain 28 parameters from two fluorescence-based techniques: flow cytometry and cell imaging. The results showed that none of the parameters alone identified gender and smoking status, but that statistical analysis of these parameters, whether or not combined with a third set of data, hematological data, can. Statistical analysis of selected parameters clearly discriminates between male and female samples, as well as smokers and non-smokers. Effects of tobacco smoke pollutants are more pronounced in female smokers than in other groups.

Altered Endoplasmic Reticulum Integrity and Organelle Interactions in Living Cells Expressing INF2 Variants.

The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER-mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

Corrigendum to "In Vivo Stem Cell Imaging Principles and Applications".

MoS2-based earth-like self-rotating and magnetically navigated nanorobots for magnetic resonance imaging, cancer cell imaging, and therapy

Microrobots are poised to revolutionize mass transport systems in emerging biomedical applications, yet their erratic motion poses a significant challenge in controlling their movements, particularly in-vivo. In this study, we introduce highly flexible, self-rotating nanorobots based on MoS2 Nanoflowers (NFs), offering superior control over their movement under magnetic field for cancer cell imaging and therapy. These nanorobots, termed MNBOTs, are constructed by embellishing pre-formed MoS2 NFs with NiFe2O3 nanoparticles (NPs) using polymeric linkers, capitalizing on the abundance of disulfide bonds on the MoS2 surface. In-vitro experiments showcased MNBOT's precise control over velocity, trajectory, and curvature, adapting seamlessly to changes in magnetic flux density under electromagnetic navigation. Moreover, MNBOTs were able to release NiFe2O3 NPs successfully in the tumor environment, facilitated by the collapse of disulfide bonds in the presence of glutathione/dithiothreitol, thus ensuring MNBOT's retrieval post-cancer therapy. Furthermore, we leveraged the photo-heat generation and paramagnetic features of MNBOTs for chemo-photothermal therapy (PTT) and magnetic resonance imaging (MRI) in ex vivo clinical settings. The combined effect of chemotherapy-PTT demonstrated remarkable cytotoxicity against MDA-MB-231 cancer cells, highlighting the synergistic potential of MNBOTs. The integration of diverse functionalities within MNBOTs, including remote magnetic navigation, photothermal therapy, and MRI, presents a versatile platform for addressing pressing healthcare needs, thus holding immense potential for future therapeutic and diagnostic applications.