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

Spatial light modulators have become an essential tool for advanced microscopy, enabling breakthroughs in 3D, phase, and super-resolution imaging. However, continuous spatial-light modulation that is capable of capturing sub-millisecond microscopic motion without diffraction artifacts and polarization dependence is challenging. Here we present a photothermal spatial light modulator (PT-SLM) enabling fast phase imaging for nanoscopic 3D reconstruction. The PT-SLM can generate a step-like wavefront change, free of diffraction artifacts, with a high transmittance and a modulation efficiency independent of light polarization. We achieve a phase-shift > π and a response time as short as 70 µs with a theoretical limit in the sub microsecond range. We used the PT-SLM to perform quantitative phase imaging of sub-diffractional species to decipher the 3D nanoscopic displacement of microtubules and study the trajectory of a diffusive microtubule-associated protein, providing insights into the mechanism of protein navigation through a complex microtubule network.

• MeSH
• časové faktory MeSH
• interferenční mikroskopie metody   statistika a číselné údaje   MeSH
• kovové nanočástice ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• mikroskopie fázově kontrastní metody   statistika a číselné údaje   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• nanotechnologie MeSH
• nanotrubičky ultrastruktura   MeSH
• optické jevy MeSH
• počítačová simulace MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny metabolismus   MeSH
• světlo MeSH
• tubulin metabolismus   MeSH
• zlato MeSH
• zobrazování trojrozměrné metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: The aim of this study was to use digital holographic microscopy (DHM) in human sperm imaging and compare quantitative phase contrast of sperm heads in normozoospermia (NZ) and oligoasthenozoospermia (OAT). METHODS: DHM spermatozoa imaging and repeated quantitative phase shift evaluation were used. Five NZ and 5 OAT samples were examined. Semen samples were examined by semen analysis and processed for DHM. Main outcome measures were maximum phase shift value of the sperm heads. Differences of the phase shift and in NZ and OAT samples were statistically tested. RESULTS: In NZ samples median phase shifts were in the range 2.72-3.21 rad and 2.00-2.15 in OAT samples. Differences among individual samples were statistically significant (p < 0.001) in both groups. Median phase shift according to sperm count was 2.90 rad in NZ samples and 2.00 rad in OAT samples. This difference was statistically significant (p < 0.001). CONCLUSION: Quantitative evaluation of the phase shift by DHM could provide new information on the exact structure and composition of the sperm head. At present, this technique is not established for clinical utility.

• MeSH
• dospělí MeSH
• hlavička spermie ultrastruktura   MeSH
• holografie MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• spermie ultrastruktura   MeSH
• Check Tag
• dospělí MeSH
• 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

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

In this paper, a novel U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance from 0.67 to 0.70 of object-wise intersection over union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

• Publikační typ
• časopisecké články 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.

• 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

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.

• 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.

• 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

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.

• MeSH
• buněčné jádro virologie   MeSH
• fluorescenční mikroskopie MeSH
• HIV infekce virologie   MeSH
• HIV-1 chemie   genetika   fyziologie   MeSH
• integrace viru MeSH
• internalizace viru * MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH