coherence-controlled holographic imaging/microscopy
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Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.
- MeSH
- buněčná adheze genetika fyziologie MeSH
- buněčné linie MeSH
- fibroblasty cytologie metabolismus MeSH
- fokální adhezní tyrosinkinasy genetika metabolismus MeSH
- krysa rodu rattus MeSH
- mikroskopie fázově kontrastní MeSH
- pohyb buněk genetika fyziologie MeSH
- polarita buněk genetika fyziologie MeSH
- receptory pro aktivovanou kinasu C genetika metabolismus MeSH
- RNA interference MeSH
- tvar buňky genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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).
- MeSH
- aktiny metabolismus MeSH
- buněčný převod mechanických signálů * MeSH
- cisplatina farmakologie MeSH
- hojení ran MeSH
- invazivní růst nádoru MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádory prostaty farmakoterapie patologie MeSH
- proliferace buněk * MeSH
- protinádorové látky farmakologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné kultury metody MeSH
- fibrosarkom diagnostické zobrazování patologie MeSH
- holografie přístrojové vybavení metody MeSH
- intravitální mikroskopie přístrojové vybavení metody MeSH
- invazivní růst nádoru diagnostické zobrazování patologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- pohyb buněk * MeSH
- pseudopodia metabolismus MeSH
- zobrazování trojrozměrné přístrojové vybavení metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the last few years, classification of cells by machine learning has become frequently used in biology. However, most of the approaches are based on morphometric (MO) features, which are not quantitative in terms of cell mass. This may result in poor classification accuracy. Here, we study the potential contribution of coherence-controlled holographic microscopy enabling quantitative phase imaging for the classification of cell morphologies. We compare our approach with the commonly used method based on MO features. We tested both classification approaches in an experiment with nutritionally deprived cancer tissue cells, while employing several supervised machine learning algorithms. Most of the classifiers provided higher performance when quantitative phase features were employed. Based on the results, it can be concluded that the quantitative phase features played an important role in improving the performance of the classification. The methodology could be valuable help in refining the monitoring of live cells in an automated fashion. We believe that coherence-controlled holographic microscopy, as a tool for quantitative phase imaging, offers all preconditions for the accurate automated analysis of live cell behavior while enabling noninvasive label-free imaging with sufficient contrast and high-spatiotemporal phase sensitivity.
- MeSH
- algoritmy MeSH
- buňky klasifikace cytologie MeSH
- holografie metody MeSH
- lidé MeSH
- mikroskopie metody MeSH
- rozpoznávání automatizované MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Coherence-controlled holographic microscopy (CCHM) in low-coherence mode possesses a pronounced coherence gate effect. This offers an option to investigate the details of cellular events leading to cell death caused by cytopathic turbid emulsions. CCHM capacity was first assessed in model situations that showed clear images obtained with low coherence of illumination but not with high coherence of illumination. Then, the form of death of human cancer cells induced by treatment with biologically active phospholipids (BAPs) preparation was investigated. The observed overall retraction of cell colony was apparently caused by the release of cell-to-substratum contacts. This was followed by the accumulation of granules decorating the nuclear membrane. Then, the occurrence of nuclear membrane indentations signaled the start of damage to the integrity of the cell nucleus. In the final stage, cells shrunk and disintegrated. This indicated that BAPs cause cell death by necrosis and not apoptosis. An intriguing option of checking the fate of cancer cells caused by the anticipated cooperative effect after adding another tested substance sodium dichloroacetate to turbid emulsion is discussed on grounds of pilot experiments. Such observations should reveal the impact and mechanism of action of the interacting drugs on cell behavior and fate that would otherwise remain hidden in turbid milieu.
- MeSH
- buněčná smrt fyziologie MeSH
- cytologické techniky metody MeSH
- fosfolipidy MeSH
- holografie metody MeSH
- kyselina dichloroctová MeSH
- lidé MeSH
- mikroskopie metody MeSH
- nádorové buněčné linie MeSH
- nádory patofyziologie MeSH
- nekróza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A coherence-controlled holographic microscope (CCHM) enables quantitative phase imaging with coherent as well as incoherent illumination. The low spatially coherent light induces a coherence gating effect, which makes observation of samples possible also through scattering media. The paper describes theoretically and simulates numerically imaging of a two-dimensional object through a static scattering layer by means of CCHM, with the main focus on the quantitative phase imaging quality. The authors have investigated both strongly and weakly scattering media characterized by different amounts of ballistic and diffuse light. It is demonstrated that the phase information can be revealed also for the case of the static, strongly scattering layer. The dependence of the quality of imaging process on the spatial light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with a model phase object, as well as living carcinoma cells treated in an optically turbid emulsion.
