Experimentální biofotonika

Browse

Now showing 1 - 5 of 40

Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis
(PLOS, 2015-03-24) Balvan, Jan; Křížová, Aneta; Gumulec, Jaromír; Raudenská, Martina; Sládek, Zbyšek; Sedláčková, Miroslava; Babula, Petr; Svobodová, Markéta; Kizek, René; Chmelík, Radim; Masařík, Michal
Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection Multimodal holographic microscopy (MHM). The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.
Addressing cancer invasion and cell motility with quantitative light microscopy
(NATURE PORTFOLIO, 2022-01-10) Zicha, Daniel
The incidence of death caused by cancer has been increasing worldwide. The growth of cancer cells is not the main problem. The majority of deaths are due to invasion and metastasis, where cancer cells actively spread from primary tumors. Our inbred rat model of spontaneous metastasis revealed dynamic phenotype changes in vitro correlating with the metastatic potential in vivo and led to a discovery of a metastasis suppressor, protein 4.1B, which affects their 2D motility on flat substrates. Subsequently, others confirmed 4.1B as metastasis suppressor using knock-out mice and patient data suggesting mechanism involving apoptosis. There is evidence that 2D motility may be differentially controlled to the 3D situation. Here we show that 4.1B affects cell motility in an invasion assay similarly to the 2D system, further supporting our original hypothesis that the role of 4.1B as metastasis suppressor is primarily mediated by its effect on motility. This is encouraging for the validity of the 2D analysis, and we propose Quantitative Phase Imaging with incoherent light source for rapid and accurate testing of cancer cell motility and growth to be of interest for personalized cancer treatment as illustrated in experiments measuring responses of human adenocarcinoma cells to selected chemotherapeutic drugs.
Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion.
(Journal of Biomedical Optics SPIE, 2015-11-01) Čolláková, Jana; Křížová, Aneta; Kollárová, Věra; Dostál, Zbyněk; Slabá, Michala; Veselý, Pavel; Chmelík, Radim
Coherence-controlled holographic microscopy (CCHM) in low-coherence mode possesses 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.
Advanced Microscopical Non-Invasive Examination of the Supposed Migrastatics for Impact on In Vitro Cell Migration
(European Conference of Oncology Pharmacy, 2022-07-02) Šuráňová, Markéta; Zábranská, Magdaléna; Kolínková, Veronika; Muchová, Nikola; Jůzová, Veronika; Chmelík, Radim; Veselý, Pavel
Live H1299 lung carcinoma cells in vitro were exposed to selected drugs with a presumed antimigration activity that implies antimetastatic potential and time-lapse examined with Coherence Controlled Holography Microscopy (CCHM) with holographic incoherent light Quantitive Phase Imaging (hiQPI). It is a methodology that online evaluates the dynamics of morphology, migration, and the growth of tumor cells by weighing them. Material and method: Q-Phase (Telight, Brno, Czech Republic) as a commercially available CCHM was employed for the hiQPI of cells. Four putative migrastatics, vincristine (VIN, 100 nM), doxycycline (DOXY, 1 mg/ml), and 4-hydroxyacetophenone (4HAP, 4 M) were tested with H1299, using Ibidi -Slide VI 0.4 for 20 hours recording with Q-Phase. Cells were cultivated at 37°C in a humidified incubator with 3.5% CO2 in standard Eagle MEM medium with 10% fetal bovine serum, 20 M gentamicin, and 2mM L-glutamine. For the time-lapse recording, the medium was enriched with 20 mM HEPES to maintain pH 7.4. Results and discussion: This research showed that on the cancer cell line H1299 the vincristine and doxycycline had the greatest migrastatic effect in the 2D environment under given conditions. These putative migrastatics showed an effect on the dynamics of migration and cell morphology. Conclusion: The hiQPI screening is a reliable and economical approach to in vitro introductory testing of potential migrastatics. hiQPI combines high-precision cell imaging, which is important for cell segmentation and thus tracking cell trajectories, with cell growth measurements, thus providing a comprehensive assessment of a potential risk of some cytopathic issues.
Single-Shot Three-Dimensional Orientation Imaging of Nanorods Using Spin to Orbital Angular Momentum Conversion
(American Chemical Society, 2021-08-25) Fordey, Tomáš; Bouchal, Petr; Schovánek, Petr; Baránek, Michal; Bouchal, Zdeněk; Viewegh, Petr; Hrtoň, Martin; Rovenská, Katarína; Ligmajer, Filip; Chmelík, Radim; Šikola, Tomáš
The key information about any nanoscale system relates to the orientations and conformations of its parts. Unfortunately, these details are often hidden below the diffraction limit, and elaborate techniques must be used to optically probe them. Here we present imaging of the 3D rotation motion of metal nanorods, restoring the distinct nanorod orientations in the full extent of azimuthal and polar angles. The nanorods imprint their 3D orientation onto the geometric phase and space-variant polarization of the light they scatter. We manipulate the light angular momentum and generate optical vortices that create self-interference images providing the nanorods’ angles via digital processing. After calibration by scanning electron microscopy, we demonstrated time-resolved 3D orientation imaging of sub-100 nm nanorods under Brownian motion (frame rate up to 500 fps). We also succeeded in imaging nanorods as nanoprobes in live-cell imaging and reconstructed their 3D rotational movement during interaction with the cell membrane (100 fps).

Browse

Recent Submissions