We demonstrate the formation of the near field with non-trivial phase distribution using surface plasmon interference devices, and experimental quantitative imaging of that phase with near-field phase microscopy. The phase distribution formed with a single device can be controlled by the polarization of the external illumination and the area of the device assigned to the object wave. A comparison of the experimental data to a numerical electromagnetic model and an analytical model assigns the origin of the near-field phase to the out-of-plane electric component of surface plasmon polaritons, and also verifies the predictive power of the models. We demonstrate a formation of near-field plane waves with different propagation directions on a single device, or even simultaneously at distinct areas of a single device. Our findings open the way to the imaging and tomography of phase objects in the near field.

• Klíčová slova
• SNOM, SPP waves, interference nanostructures, near-field, phase imaging,
• Publikační typ
• časopisecké články MeSH

SIGNIFICANCE: Machine learning is increasingly being applied to the classification of microscopic data. In order to detect some complex and dynamic cellular processes, time-resolved live-cell imaging might be necessary. Incorporating the temporal information into the classification process may allow for a better and more specific classification. AIM: We propose a methodology for cell classification based on the time-lapse quantitative phase images (QPIs) gained by digital holographic microscopy (DHM) with the goal of increasing performance of classification of dynamic cellular processes. APPROACH: The methodology was demonstrated by studying epithelial-mesenchymal transition (EMT) which entails major and distinct time-dependent morphological changes. The time-lapse QPIs of EMT were obtained over a 48-h period and specific novel features representing the dynamic cell behavior were extracted. The two distinct end-state phenotypes were classified by several supervised machine learning algorithms and the results were compared with the classification performed on single-time-point images. RESULTS: In comparison to the single-time-point approach, our data suggest the incorporation of temporal information into the classification of cell phenotypes during EMT improves performance by nearly 9% in terms of accuracy, and further indicate the potential of DHM to monitor cellular morphological changes. CONCLUSIONS: Proposed approach based on the time-lapse images gained by DHM could improve the monitoring of live cell behavior in an automated fashion and could be further developed into a tool for high-throughput automated analysis of unique cell behavior.

• Klíčová slova
• digital holographic microscopy, epithelial–mesenchymal transition, quantitative phase imaging, supervised machine learning,
• MeSH
• algoritmy MeSH
• časosběrné zobrazování MeSH
• epitelo-mezenchymální tranzice * MeSH
• holografie * MeSH
• strojové učení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• Rack1, cell adhesion, cell dry mass, cell spreading, coherence-controlled holographic microscopy, extracellular matrix, focal adhesion kinase, front–rear polarity, quantitative phase imaging,
• 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
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• RACK1 protein, rat MeSH Prohlížeč
• receptory pro aktivovanou kinasu C MeSH

Cell viability and cytotoxicity assays are highly important for drug screening and cytotoxicity tests of antineoplastic or other therapeutic drugs. Even though biochemical-based tests are very helpful to obtain preliminary preview, their results should be confirmed by methods based on direct cell death assessment. In this study, time-dependent changes in quantitative phase-based parameters during cell death were determined and methodology useable for rapid and label-free assessment of direct cell death was introduced. The goal of our study was distinction between apoptosis and primary lytic cell death based on morphologic features. We have distinguished the lytic and non-lytic type of cell death according to their end-point features (Dance of Death typical for apoptosis versus swelling and membrane rupture typical for all kinds of necrosis common for necroptosis, pyroptosis, ferroptosis and accidental cell death). Our method utilizes Quantitative Phase Imaging (QPI) which enables the time-lapse observation of subtle changes in cell mass distribution. According to our results, morphological and dynamical features extracted from QPI micrographs are suitable for cell death detection (76% accuracy in comparison with manual annotation). Furthermore, based on QPI data alone and machine learning, we were able to classify typical dynamical changes of cell morphology during both caspase 3,7-dependent and -independent cell death subroutines. The main parameters used for label-free detection of these cell death modalities were cell density (pg/pixel) and average intensity change of cell pixels further designated as Cell Dynamic Score (CDS). To the best of our knowledge, this is the first study introducing CDS and cell density as a parameter typical for individual cell death subroutines with prediction accuracy 75.4% for caspase 3,7-dependent and -independent cell death.

• MeSH
• algoritmy MeSH
• apoptóza * účinky léků   MeSH
• buněčná smrt * účinky léků   MeSH
• buňky ultrastruktura   MeSH
• časosběrné zobrazování metody   MeSH
• časové faktory MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• optické zobrazování metody   MeSH
• počet buněk MeSH
• statistické modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: 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. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

• Klíčová slova
• Cell segmentation, Differential contrast image, Image reconstruction, Laplacian of Gaussians, Methods comparison, Microscopy, Quantitative phase imaging,
• MeSH
• algoritmy MeSH
• frakcionace buněk metody   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• počítačové zpracování obrazu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

We present geometric-phase microscopy allowing a multipurpose quantitative phase imaging in which the ground-truth phase is restored by quantifying the phase retardance. The method uses broadband spatially incoherent light that is polarization sensitively controlled through the geometric (Pancharatnam-Berry) phase. The assessed retardance possibly originates either in dynamic or geometric phase and measurements are customized for quantitative mapping of isotropic and birefringent samples or multi-functional geometric-phase elements. The phase restoration is based on the self-interference of polarization distinguished waves carrying sample information and providing pure reference phase, while passing through an inherently stable common-path setup. The experimental configuration allows an instantaneous (single-shot) phase restoration with guaranteed subnanometer precision and excellent ground-truth accuracy (well below 5 nm). The optical performance is demonstrated in advanced yet routinely feasible noninvasive biophotonic imaging executed in the automated manner and predestined for supervised machine learning. The experiments demonstrate measurement of cell dry mass density, cell classification based on the morphological parameters and visualization of dynamic dry mass changes. The multipurpose use of the method was demonstrated by restoring variations in the dynamic phase originating from the electrically induced birefringence of liquid crystals and by mapping the geometric phase of a space-variant polarization directed lens.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Artifact-free microscopic images represent a key requirement of multi-parametric image analysis in modern biomedical research. Holography microscopy (HM) is one of the quantitative phase imaging techniques, which has been finding new applications in life science, especially in morphological screening, cell migration, and cancer research. Rather than the classical imaging of absorbing (typically stained) specimens by bright-field microscopy, the information about the light-wave's phase shifts induced by the biological sample is employed for final image reconstruction. In this comparative study, we investigated the usability and the reported advantage of the holography imaging. The claimed halo-free imaging was analyzed compared to the widely used Zernike phase-contrast microscopy. The intensity and phase cross-membrane profiles at the periphery of the cell were quantified. The intensity profile for cells in the phase-contrast images suffers from the significant increase in intensity values around the cell border. On the contrary, no distorted profile is present outside the cell membrane in holography images. The gradual increase in phase shift values is present in the internal part of the cell body projection in holography image. This increase may be related to the increase in the cell internal material according to the dry mass theory. Our experimental data proved the halo-free nature of the holography imaging, which is an important prerequisite of the correct thresholding and cell segmentation, nowadays frequently required in high-content screening and other image-based analysis. Consequently, HM is a method of choice whenever the image analysis relies on the accurate data on cell boundaries.

• Klíčová slova
• Halo effect, Holography microscopy, Phase-contrast,
• MeSH
• artefakty MeSH
• HeLa buňky MeSH
• holografie * MeSH
• lidé MeSH
• mikroskopie fázově kontrastní * MeSH
• nádorové buňky kultivované MeSH
• Saccharomyces cerevisiae cytologie   růst a vývoj   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

• MeSH
• analýza hlavních komponent MeSH
• autofagie účinky léků   MeSH
• buněčná sebeobnova * účinky léků   MeSH
• časosběrné zobrazování MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• entóza účinky léků   MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• metastázy nádorů MeSH
• mezibuněčná komunikace účinky léků   MeSH
• mitofagie účinky léků   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty genetika   patologie   MeSH
• naftochinony farmakologie   MeSH
• oxidační stres * účinky léků   MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• velikost buňky účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• naftochinony MeSH
• plumbagin MeSH Prohlížeč
• reaktivní formy kyslíku MeSH

A coherence-controlled holographic microscope (CCHM) was developed particularly for quantitative phase imaging and measurement of live cell dynamics, which is the proper subject of digital holographic microscopy (DHM). CCHM in low-coherence mode extends DHM in the study of living cells. However, this advantage is compensated by sensitivity of the system to easily become misaligned, which is a serious hindrance to wanted performance. Therefore, it became clear that introduction of a self-correcting system is inevitable. Accordingly, we had to devise a theory of a suitable control and design an automated alignment system for CCHM. The modulus of the reconstructed holographic signal was identified as a significant variable for guiding the alignment procedures. From this, we derived the original basic realignment three-dimensional algorithm, which encompasses a unique set of procedures for automated alignment that contains processes for initial and advanced alignment as well as long-term maintenance of microscope tuning. All of these procedures were applied to a functioning microscope and the tested processes were successfully validated. Finally, in such a way, CCHM is enabled to substantially contribute to study of biology, particularly of cancer cells in vitro.

• Klíčová slova
• holographic microscopy *,
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• apoptóza * MeSH
• časové faktory MeSH
• fenotyp MeSH
• fluorescenční mikroskopie metody   MeSH
• holografie metody   MeSH
• lidé MeSH
• multimodální zobrazování metody   MeSH
• nádorové buněčné linie MeSH
• nekróza MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH