Cell segmentation methods for label-free contrast microscopy: review and comprehensive comparison

Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities

Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light

Increased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm2, 208 mJ/cm2 and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm2 × 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm2 × 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition

Complexes of Silver(I) Ions and Silver Phosphate Nanoparticles with Hyaluronic Acid and/or Chitosan as Promising Antimicrobial Agents for Vascular Grafts

Polymers are currently widely used to replace a variety of natural materials with respect to their favourable physical and chemical properties, and due to their economic advantage. One of the most important branches of application of polymers is the production of different products for medical use. In this case, it is necessary to face a significant disadvantage of polymer products due to possible and very common colonization of the surface by various microorganisms that can pose a potential danger to the patient. One of the possible solutions is to prepare polymer with antibacterial/antimicrobial properties that is resistant to bacterial colonization. The aim of this study was to contribute to the development of antimicrobial polymeric material ideal for covering vascular implants with subsequent use in transplant surgery. Therefore, the complexes of polymeric substances (hyaluronic acid and chitosan) with silver nitrate or silver phosphate nanoparticles were created, and their effects on gram-positive bacterial culture of Staphylococcus aureus were monitored. Stages of formation of complexes of silver nitrate and silver phosphate nanoparticles with polymeric compounds were characterized using electrochemical and spectrophotometric methods. Furthermore, the antimicrobial activity of complexes was determined using the methods of determination of growth curves and zones of inhibition. The results of this study revealed that the complex of chitosan, with silver phosphate nanoparticles, was the most suitable in order to have an antibacterial effect on bacterial culture of Staphylococcus aureus. Formation of this complex was under way at low concentrations of chitosan. The results of electrochemical determination corresponded with the results of spectrophotometric methods and verified good interaction and formation of the complex. The complex has an outstanding antibacterial effect and this effect was of several orders higher compared to other investigated complexes

Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation

We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935)

Cellular mechanosensing by means of atomic force microscopy

Mechanobiological sensing brings together biology, physics, medicine and engineering, thus helps to characterize how the protein molecules, cells and tissues respond to mechanical cues contribute to differentiation, development, structural and disease processes. The mechanobiology contributes to recognition of the sensing, transduction and application of mechanical signals by the biological systems. Atomic force microscopy (AFM) has grew up from the solid material characterization method to the a important device allowing the simultaneous topographical and mechanical characterization of living biological systems. In this work such a potential of the AFM method will be described on selected examples. It was shown that cell stiffness determined by AFM can be used as a marker for cancer progression and metastatic potential. Different cancer types feature distinct cell stiffness and a connection between attenuated cell stiffness and increased invasion capacity was also observed. The force microscope can serve as mechanotransducing actuator of the cardiac cells contractility. Combination with the other methods, such as microelectrode array, leads to a comprehensive description of the contractile phenomenon. Pathophysiological electro-mechanical coupling needs to be characterized in a detail, if the alterations often resulting in mechanical heart failure would be understand and treated. \We would like to demonstrate AFM together with other biophysical methods brings a promising approach that helps understand the correlation between the cell structure, cell mechanics, and function

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGF ss 1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-a, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators

Addition of platelet concentrate to Dermo-Epidermal Skin Graft in deep burn trauma reduces scarring and need for revision surgeries

Backround. Deep skin burn injuries, especially those on the face, hands, feet, genitalia and perineum represent significant therapeutic challenges. Autologous dermo-epidermal skin grafts (DESG) have become standard of care for treating deep burns. Additionally, human autologous thrombin activated autologous platelet concentrate (APC) has gained acceptance in the setting of wounds. While each of these interventions has been independently shown to accelerate healing, the combination of the two has never been evaluated. We hypothesized that the addition of platelets (source of growth factors and inhibitors necessary for tissue repair) to the DESG (source of progenitor cells and of tissue proteases necessary for spatial and temporal control of growth regulators released from platelets) would create the optimal environment for the reciprocal interaction of cells within the healing tissues. Methods: We used clinical examination (digital photography), standardised scales for evaluating pain and scarring, in combination with blood perfusion (laser Doppler imaging), as well as molecular and laboratory analyses. Results: We show for the first time that the combination of APC and DESG leads to earlier relief of pain, and decreased use of analgesics, antipruritics and orthotic devices. Most importantly, this treatment is associated with earlier discharges from hospital and significant cost savings. Conclusions: Our findings indicate that DESG engraftment is facilitated by the local addition of platelets and by systemic thrombocytosis. This local interaction leads to the physiological revascularization at 1-3 months. We observed significant elevation of circulating platelets in early stages of engraftment (1-7 days), which normalized over the subsequent 7 and 90 days.Web of Science158225824

Modulation of Induced Cytotoxicity of Doxorubicin by Using Apoferritin and Liposomal Cages

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin-and liposome-encapsulated forms of doxorubicin ("Apodox" and "lip-8-dox") and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP