Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope.

• MeSH
• algoritmy MeSH
• fibrosarkom patologie   MeSH
• holografie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• krysa rodu rattus MeSH
• mikroskopie fázově kontrastní metody   MeSH
• nádorové buněčné linie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata 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.

• MeSH
• algoritmy MeSH
• holografie metody   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• nádorové buněčné linie MeSH
• počítačová simulace MeSH
• počítačové zpracování obrazu metody   MeSH
• radiační rozptyl MeSH
• světlo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Head and neck squamous cell carcinoma is one of the most aggressive tumours and is typically diagnosed too late. Late diagnosis requires an urgent decision on an effective therapy. An individualized test of chemosensitivity should quickly indicate the suitability of chemotherapy and radiotherapy. No ex vivo chemosensitivity assessment developed thus far has become a part of general clinical practice. Therefore, we attempted to explore the new technique of coherence-controlled holographic microscopy to investigate the motility and growth of live cells from a head and neck squamous cell carcinoma biopsy. We expected to reveal behavioural patterns characteristic for malignant cells that can be used to imrove future predictive evaluation of chemotherapy. We managed to cultivate primary SACR2 carcinoma cells from head and neck squamous cell carcinoma biopsy verified through histopathology. The cells grew as a cohesive sheet of suspected carcinoma origin, and western blots showed positivity for the tumour marker p63 confirming cancerous origin. Unlike the roundish colonies of the established FaDu carcinoma cell line, the SACR2 cells formed irregularly shaped colonies, eliciting the impression of the collective invasion of carcinoma cells. Time-lapse recordings of the cohesive sheet activity revealed the rapid migration and high plasticity of these epithelial-like cells. Individual cells frequently abandoned the swiftly migrating crowd by moving aside and crawling faster. The increasing mass of fast migrating epithelial-like cells before and after mitosis confirmed the continuation of the cell cycle. In immunofluorescence, analogously shaped cells expressed the p63 tumour marker, considered proof of their origin from a carcinoma. These behavioural traits indicate the feasible identification of carcinoma cells in culture according to the proposed concept of the carcinoma cell dynamic phenotype. If further developed, this approach could later serve in a new functional online analysis of reactions of carcinoma cells to therapy. Such efforts conform to current trends in precision medicine.

• MeSH
• biopsie MeSH
• buněčný cyklus fyziologie   MeSH
• holografie metody   MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• nádory hlavy a krku metabolismus   patologie   MeSH
• pohyb buněk fyziologie   MeSH
• senioři MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři 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

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

• MeSH
• buněčné linie MeSH
• cytologické techniky metody   MeSH
• holografie metody   MeSH
• krysa rodu rattus MeSH
• mikroskopie metody   MeSH
• osmotický tlak fyziologie   MeSH
• tvar buňky fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

• MeSH
• apoptóza MeSH
• B-lymfocyty cytologie   MeSH
• buněčný cyklus MeSH
• checkpoint kinasa 1 antagonisté a inhibitory   MeSH
• chronická lymfatická leukemie farmakoterapie   genetika   metabolismus   MeSH
• cytarabin aplikace a dávkování   MeSH
• deoxycytidin aplikace a dávkování   analogy a deriváty   MeSH
• lidé MeSH
• mitóza MeSH
• mutace MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   MeSH
• nukleosidy genetika   MeSH
• proliferace buněk MeSH
• pyrazoly farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• signální transdukce MeSH
• viabilita buněk MeSH
• vidarabin aplikace a dávkování   analogy a deriváty   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH