In this work, we classify chemotherapeutic agents (topoisomerase inhibitors) based on their effect on U-2 OS cells. We use phase-contrast microscopy images, which are faster and easier to obtain than fluorescence images and support live cell imaging. We use a convolutional neural network (CNN) trained end-to-end directly on the input images without requiring for manual segmentations or any other auxiliary data. Our method can distinguish between tested cytotoxic drugs with an accuracy of 98%, provided that their mechanism of action differs, outperforming previous work. The results are even better when substance-specific concentrations are used. We show the benefit of sharing the extracted features over all classes (drugs). Finally, a 2D visualization of these features reveals clusters, which correspond well to known class labels, suggesting the possible use of our methodology for drug discovery application in analyzing new, unseen drugs.
Optická koherentní tomografie (OCT) s rozlišením 10-15 μm je v současné době nejpřesnější zobrazovací modalita invazivního vyšetřování koronárních tepen. S novými možnostmi zpracování signálů a obrazů a využitím umělé inteligence v medicíně postupuje i evoluce této metody kupředu. Článek popisuje historii vývoje OCT, technické detaily této modality a shrnuje nejnovější data, kterých bylo dosaženo ve vědeckém výzkumu. Zavedení nových softwarů a hybridních modalit v kardiologii může přinést nové možnosti v kvantitativním a kvalitativním hodnocení koronárních tepen během intervenčního výkonu.
Optical coherence tomography (OCT) with a resolution of 10-15 μm is currently the most accurate imaging modality for invasive coronary artery evaluation. As the options of signal and image processing and of using artificial intelligence in medicine progress, so does the evolution of OCT imaging. The article describes the history of OCT development and technical details of this modality, and summarizes the latest data obtained in scientific research. The introduction of novel software tools and hybrid modalities in cardiology may bring new possibilities in quantitative and qualitative assessment of coronary arteries during an interventional procedure.
- MeSH
- Interferometry MeSH
- Coronary Vessels diagnostic imaging MeSH
- Humans MeSH
- Tomography, Optical Coherence * history methods trends MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Simultaneous application of polarization microscopy and Interphako interference contrast has been used to study the internal structure of algal cells. The interference contrast technique showed fine cell structures (important is the selection of interference colors according to the Mach-Zehnder interferometer setting). In a polarization microscope, the crossed polarization filters together with the first-order quartz compensator mounted turntable showed the maximum birefringence of the individual structures. Material containing green algae was collected in the villages Sýkořice and Zbečno, Protected Landscape Area (PLA) Křivoklátsko. The objects were studied in a Carl Zeiss Jena NfpK laboratory microscope equipped with an In 160 base body with an Interphako In contrast interference module including a Mach-Zehnder interferometer with variable phase contrast, a special condenser with interchangeable aperture plates, a turntable, a Meopta Praha polarizer, a LOMO Sankt Petersburg analyzer, and a quartz compensator with first-order red and the digital camera DSLR Nikon D 70. Green algae of three orders were studied: Siphonocladales, Zygnematales, and Desmidiales. Anisotropic structures were found in all studied representatives of the green algae of the phylum Chlorophyta. Especially their cell walls showed strong birefringence (in all representatives of these orders). On the other hand, a representative of the order Siphonocladales (the genus Cladophora, Cladophoraceae, Ulvophyceae) was rarely found to display weak birefringent granules of storage substances due to the setting of the Mach-Zehnder interferometer and the use of the first-order compensator (interference colors are intensified). In addition, a very weak birefringence of periphyton cells (microbial biofilm) was found. In the study of the second algae of the genus Spirogyra (Zygnemataceae, Zygnematales, Conjugatophyceae), a strongly birefringent connecting wall between algal cells was found in contrast to the weaker birefringence of the peripheral wall. It was the use of Interphako interference contrast together with polarization filters and a first-order quartz compensator that particularly emphasized the central part of the connecting wall. In the study of the twinned Pleurotaenium algae (Desmidiaceae, Desmidiales, Conjugatophyceae), a strongly birefringent wall was found along the periphery of the cell with a nucleus in the middle part (isthmus). In this narrowing in the center of the cell, a sharply delimited birefringent edge of the cell wall is visible, especially when using Interphako interference contrast along with crossed polarization filters and a first-order quartz compensator. In conclusion, Interphako interference contrast provides a high degree of image contrast in a microscope and, if suitably simultaneously complemented by polarization microscopy (including a first-order quartz compensator), it will allow us to infer some of the composition of the investigated structures. However, working with Interphako interference contrast is considerably more difficult (setting Mach-Zehnder interferometer) than using other contrast techniques (positive and negative phase contrast, color contrast, relief contrast, and dark field).
- MeSH
- Chlorophyta * MeSH
- Birefringence MeSH
- Microscopy, Phase-Contrast MeSH
- Microscopy, Polarization MeSH
- Zygnematales * MeSH
- Publication type
- Journal Article MeSH
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
- MeSH
- Adenosine Diphosphate pharmacology MeSH
- Adenosine Triphosphate pharmacology MeSH
- Caenorhabditis elegans metabolism MeSH
- Species Specificity MeSH
- Fluorescence MeSH
- Interferometry MeSH
- DNA, Single-Stranded metabolism MeSH
- Nucleotides metabolism MeSH
- Caenorhabditis elegans Proteins metabolism MeSH
- Rad51 Recombinase chemistry metabolism MeSH
- Sequence Homology, Amino Acid * MeSH
- Protein Stability drug effects MeSH
- Protein Binding drug effects MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Time Factors MeSH
- Microscopy, Interference methods statistics & numerical data MeSH
- Metal Nanoparticles ultrastructure MeSH
- Humans MeSH
- Microscopy, Atomic Force MeSH
- Microscopy, Phase-Contrast methods statistics & numerical data MeSH
- Microtubules metabolism ultrastructure MeSH
- Nanotechnology MeSH
- Nanotubes ultrastructure MeSH
- Optical Phenomena MeSH
- Computer Simulation MeSH
- Microtubule-Associated Proteins metabolism MeSH
- Cell Cycle Proteins metabolism MeSH
- Schizosaccharomyces pombe Proteins metabolism MeSH
- Light MeSH
- Tubulin metabolism MeSH
- Gold MeSH
- Imaging, Three-Dimensional methods statistics & numerical data MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.
- MeSH
- Adaptor Proteins, Signal Transducing chemistry genetics metabolism MeSH
- Cell Membrane metabolism MeSH
- Glycine genetics metabolism MeSH
- Microscopy, Interference MeSH
- Jurkat Cells MeSH
- Microscopy, Confocal MeSH
- Humans MeSH
- Membrane Proteins chemistry genetics metabolism MeSH
- Mutation MeSH
- Proline genetics metabolism MeSH
- Protein Domains MeSH
- Protein Structure, Secondary MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Molecular Dynamics Simulation MeSH
- T-Lymphocytes metabolism MeSH
- Calcium metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cíl: Cílem této práce je porovnání měřených parametrů při použití nového biometru Argos s konvenčně užívaným biometrem IOL Master 700. Soubor a metodika: Retrospektivní analýza biometrických dat u 57 pacientů (106 očí) vyšetřených na oční klinice TANA s.r.o., v Olomouci. Měření pacienta bylo prováděno na přístrojích Argos a IOL Master 700 v jeden den stejným optometristou v rámci standardní předoperační kalkulace nitrooční čočky před operací katarakty. Hodnocenými parametry byla axiální délka bulbu, hloubka přední komory, průměrná keratometrie a tloušťka čočky, které byly následně statisticky analyzovány. Výsledky: Korelace mezi všemi porovnávanými daty byla vysoká, se signifikancí p < 0,01. Při analyzování Bland-Altmanova grafu byla nalezena dobrá shoda měření při limitu shody 95 %. Axiální délky, průměrné keratometrie a tloušťky čočky nevykazovaly signifikantní rozdíl (p = 0,941; p = 0,773; p = 0,860). Průměrné keratometrie získané biometrem IOL Master 700 byly plošší, rozdíl byl ale malý a nesignifikantní. Signifikantně větší hloubky přední komory (p < 0,05) byly zaznamenány při měření přístrojem Argos. Závěr: Technologie segmentovaného refrakčního indexu, která je využívána přístrojem Argos, zapříčinila rozdíl v hodnotách hloubky přední komory. Porovnávaná celková axiální délka ale nebyla tímto v našem souboru pacientů ovlivněna. Optické biometry Argos a IOL Master 700 celkově vykazují vynikající shodu v měřených biometrických parametrech.
Purpose: To compare the biometric data obtained by the new optical biometer Argos and the conventionally used biometer IOL Master 700. Patients and methods: Retrospective analysis of the biometric data of 57 patients (106 eyes) who were examined at TANA Ophthalmology Clinic s.r.o in Olomouc. Patient measurements were carried out on both devices on the same day by the same optometrist within the standard preoperative calculation of the intraocular lens before cataract surgery. Evaluated and statistically analyzed biometric data were axial lenght, anterior chamber depth, average keratometry and lens thickness. Results: The correlation between all compared data was high, with statistical significance p < 0.01. Bland-Altman plots showed good agreement with a 95% limit of agreement. Axial lenght, average keratometry and lens thickens did not show significant differences (p = 0.941; p = 0.773; p = 0.860). IOL Master 700 showed flatter average keratometry, however, the differences were numerically small and insignificant. Anterior chamber depths obtained by Argos were longer, with a significance p < 0.05. Conclusion: The segmental refractive index technology used by Argos caused differences in anterior chamber depths. Overall axial length was, however, not, in our cohort of patients, affected by this. In general, the optical biometers Argos and IOL Master 700 show excellent agreement in the measured biometric data.
- Keywords
- Argos,
- MeSH
- Axial Length, Eye MeSH
- Biometry * methods instrumentation MeSH
- Interferometry MeSH
- Cataract diagnosis MeSH
- Humans MeSH
- Lenses, Intraocular * MeSH
- Tomography, Optical Coherence MeSH
- Reproducibility of Results MeSH
- Retrospective Studies MeSH
- Endothelium, Corneal MeSH
- Check Tag
- Humans 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
- Cell Adhesion genetics physiology MeSH
- Cell Line MeSH
- Fibroblasts cytology metabolism MeSH
- Focal Adhesion Protein-Tyrosine Kinases genetics metabolism MeSH
- Rats MeSH
- Microscopy, Phase-Contrast MeSH
- Cell Movement genetics physiology MeSH
- Cell Polarity genetics physiology MeSH
- Receptors for Activated C Kinase genetics metabolism MeSH
- RNA Interference MeSH
- Cell Shape genetics physiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic were screened for Cryptosporidium by microscopy and PCR/sequencing at loci coding small-subunit rRNA, Cryptosporidium oocyst wall protein, actin and 70 kDa heat shock protein. Cryptosporidium infections were detected in 74 voles (22.6%). Rates of infection did not differ between males and females nor between juveniles and adults. Phylogenetic analysis revealed the presence of eight Cryptosporidium species/genotypes including two new species, C. alticolis and C. microti. These species from wild-caught common voles were able to infect common and meadow voles under experimental conditions, with a prepatent period of 3-5 days post-infection (DPI), but they were not infectious for various other rodents or chickens. Meadow voles lost infection earlier than common voles (11-14 vs 13-16 DPI) and had significantly lower infection intensity. Cryptosporidium alticolis infects the anterior small intestine and has larger oocysts (5.4 × 4.9 µm), whereas C. microti infects the large intestine and has smaller oocysts (4.3 × 4.1 µm). None of the rodents developed clinical signs of infection. Genetic and biological data support the establishment of C. alticolis and C. microti as separate species of the genus Cryptosporidium.
- MeSH
- Arvicolinae parasitology MeSH
- Cryptosporidium classification genetics ultrastructure MeSH
- Feces parasitology MeSH
- Microscopy, Fluorescence MeSH
- Phylogeny MeSH
- Gastrointestinal Tract parasitology pathology ultrastructure MeSH
- Genetic Variation MeSH
- Microscopy, Interference MeSH
- Cryptosporidiosis epidemiology parasitology transmission MeSH
- Rats MeSH
- Chickens MeSH
- Microscopy, Electron, Scanning MeSH
- Murinae MeSH
- Mice, Inbred BALB C MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Rodent Diseases epidemiology parasitology transmission MeSH
- Polymerase Chain Reaction MeSH
- Prevalence MeSH
- DNA, Protozoan chemistry genetics isolation & purification MeSH
- RNA, Ribosomal genetics MeSH
- Base Sequence MeSH
- Sequence Alignment veterinary MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Geographicals
- Czech Republic 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
- Artifacts MeSH
- HeLa Cells MeSH
- Holography * MeSH
- Humans MeSH
- Microscopy, Phase-Contrast * MeSH
- Tumor Cells, Cultured MeSH
- Saccharomyces cerevisiae cytology growth & development MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
