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.

• Klíčová slova
• Anti-cancer drugs, Convolutional neural networks, Deep learning, Drug discovery, Phase-contrast images,
• MeSH
• buněčné kultury * MeSH
• mikroskopie fázově kontrastní MeSH
• neuronové sítě (počítačové) * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• adenosindifosfát farmakologie   MeSH
• adenosintrifosfát farmakologie   MeSH
• Caenorhabditis elegans metabolismus   MeSH
• druhová specificita MeSH
• fluorescence MeSH
• interferometrie MeSH
• jednovláknová DNA metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny Caenorhabditis elegans metabolismus   MeSH
• rekombinasa Rad51 chemie   metabolismus   MeSH
• sekvenční homologie aminokyselin * MeSH
• stabilita proteinů účinky léků   MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• jednovláknová DNA MeSH
• nukleotidy MeSH
• proteiny Caenorhabditis elegans MeSH
• rekombinasa Rad51 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.

• Klíčová slova
• LAT, MD simulations, T cells, microscopy, transmembrane domain,
• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• glycin genetika   metabolismus   MeSH
• interferenční mikroskopie MeSH
• Jurkat buňky MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• mutace MeSH
• prolin genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• T-lymfocyty metabolismus   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• glycin MeSH
• LAT protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• prolin MeSH
• vápník 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
• Názvy látek
• Ase1 protein, S pombe MeSH Prohlížeč
• PRC1 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• tubulin MeSH
• zlato 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
• dvojitý lom MeSH
• mikroskopie fázově kontrastní MeSH
• polarizační mikroskopie MeSH
• Zygnematales * MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Argos, SS-OCT, biometry, segmental refractive index,
• MeSH
• axiální délka oka MeSH
• biometrie MeSH
• interferometrie MeSH
• katarakta * diagnóza   MeSH
• lidé MeSH
• nitrooční čočky * MeSH
• optická koherentní tomografie MeSH
• přední komora oční MeSH
• reprodukovatelnost výsledků MeSH
• retrospektivní studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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

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.

• Klíčová slova
• Experimental infection, Rodentia, molecular analyses, oocyst size, phylogeny, voles,
• MeSH
• Arvicolinae parazitologie   MeSH
• Cryptosporidium klasifikace   genetika   ultrastruktura   MeSH
• feces parazitologie   MeSH
• fluorescenční mikroskopie MeSH
• fylogeneze MeSH
• gastrointestinální trakt parazitologie   patologie   ultrastruktura   MeSH
• genetická variace MeSH
• interferenční mikroskopie MeSH
• kryptosporidióza epidemiologie   parazitologie   přenos   MeSH
• krysa rodu Rattus MeSH
• kur domácí MeSH
• mikroskopie elektronová rastrovací MeSH
• Murinae MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nemoci hlodavců epidemiologie   parazitologie   přenos   MeSH
• polymerázová řetězová reakce MeSH
• prevalence MeSH
• protozoální DNA chemie   genetika   izolace a purifikace   MeSH
• RNA ribozomální genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení veterinární   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• protozoální DNA MeSH
• RNA ribozomální MeSH

The publication presents a comparative study of two fibre-optic sensors in the application of heart rate (HR) and respiratory rate (RR) monitoring of the human body. After consultation with clinical practitioners, two types of non-invasive measuring and analysis systems based on fibre Bragg grating (FBG) and fibre-optic interferometer (FOI) have been designed and assembled. These systems use probes (both patent pending) that have been encapsulated in the bio-compatible polydimethylsiloxane (PMDS). The main advantage of PDMS is that it is electrically non-conductive and, as well as optical fibres, has low permeability. The initial verification measurement of the system designed was performed on four subjects in a harsh magnetic resonance (MR) environment under the supervision of a senior radiology assistant. A follow-up comparative study was conducted, upon a consent of twenty volunteers, in a laboratory environment with a minimum motion load and discussed with a head doctor of the Radiodiagnostic Institute. The goal of the laboratory study was to perform measurements that would simulate as closely as possible the environment of harsh MR or the environment of long-term health care facilities, hospitals and clinics. Conventional HR and RR measurement systems based on ECG measurements and changes in the thoracic circumference were used as references. The data acquired was compared by the objective Bland⁻Altman (B⁻A) method and discussed with practitioners. The results obtained confirmed the functionality of the designed probes, both in the case of RR and HR measurements (for both types of B⁻A, more than 95% of the values lie within the ±1.96 SD range), while demonstrating higher accuracy of the interferometric probe (in case of the RR determination, 95.66% for the FOI probe and 95.53% for the FBG probe, in case of the HR determination, 96.22% for the FOI probe and 95.23% for the FBG probe).

• Klíčová slova
• Bragg grating, ballistocardiography (BCG), biomedical engineering, electrocardiography (ECG), heart rate (HR), interferometer, magnetic resonance imaging (MRI), non-invasive measurements, patient monitoring, phonocardiography (PCG), polydimethylsiloxane (PDMS), respiratory rate (RR), vital signs,
• MeSH
• artefakty MeSH
• dechová frekvence fyziologie   MeSH
• dospělí MeSH
• elektrokardiografie MeSH
• fonokardiografie MeSH
• interferometrie přístrojové vybavení   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské tělo MeSH
• magnetická rezonanční tomografie * MeSH
• mladý dospělý MeSH
• optická vlákna MeSH
• optické jevy * MeSH
• počítačové zpracování signálu MeSH
• pohyb těles MeSH
• srdeční frekvence fyziologie   MeSH
• technologie optických vláken přístrojové vybavení   MeSH
• vlnková analýza MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkořice and Zbečno (Protected Landscape Area Křivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.

• MeSH
• anizotropie MeSH
• biologie buňky přístrojové vybavení   MeSH
• buněčná stěna chemie   MeSH
• Chlorophyta chemie   cytologie   MeSH
• cytologické techniky metody   MeSH
• cytoplazma chemie   MeSH
• dvojitý lom MeSH
• Euglenida chemie   cytologie   MeSH
• mikroskopie fázově kontrastní * MeSH
• polarizační mikroskopie * MeSH
• rozsivky chemie   cytologie   MeSH
• Zygnematales chemie   cytologie   MeSH
• Publikační typ
• časopisecké články MeSH