Advanced microscopy
Dotaz
Zobrazit nápovědu
Mitotic cell division in plants is a dynamic process playing a key role in plant morphogenesis, growth, and development. Since progress of mitosis is highly sensitive to external stresses, documentation of mitotic cell division in living plants requires fast and gentle live-cell imaging microscopy methods and suitable sample preparation procedures. This chapter describes, both theoretically and practically, currently used advanced microscopy methods for the live-cell visualization of the entire process of plant mitosis. These methods include microscopy modalities based on spinning disk, Airyscan confocal laser scanning, structured illumination, and light-sheet bioimaging of tissues or whole plant organs with diverse spatiotemporal resolution. Examples are provided from studies of mitotic cell division using microtubule molecular markers in the model plant Arabidopsis thaliana, and from deep imaging of mitotic microtubules in robust plant samples, such as legume crop species Medicago sativa.
FASEB journal. 2, ISSN 0892-6638 Supplement vol. 13
S179-S283 s. ; 30 cm
... Kreft (Ljubjana) -- 14.00 -14.20 Rescuing real time three-dimensional scanning electron microscopy (L7 ... ... Peychl (Dresden) -- 15.00-15.20 Advanced optical micromanipulation devices for bioapplications (L10) ... ... Brno) -- 15.20-15.30 Visualisation of kidney glomeruli in real time video-rate confocal reflection microscopy ...
42 s. ; 30 cm
1st ed. xii, 368 s., obr.
Advanced methods
2nd ed. xvii, 277 s. : il.
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.
Every day, genomes are affected by genotoxic factors that create multiple DNA lesions. Several DNA repair systems have evolved to counteract the deleterious effects of DNA damage. These systems include a set of DNA repair mechanisms, damage tolerance processes, and activation of cell-cycle checkpoints. This study describes selected confocal microscopy techniques that investigate DNA damage-related nuclear events after UVA- and γ-irradiation and compare the DNA damage response (DDR) induced by the two experimental approaches. In both cases, we observed induction of the nucleotide excision repair (NER) pathway and formation of localized double-strand breaks (DSBs). This was confirmed by analysis of cyclobutane pyrimidine dimers (CPDs) in the DNA lesions and by increased levels of γH2AX and 53BP1 proteins in the irradiated genome. DNA damage by UVA-lasers was potentiated by either BrdU or Hoechst 33342 pre-sensitization and compared to non-photosensitized cells. DSBs were also induced without BrdU or Hoechst 33342 pre-treatment. Interestingly, no cyclobutane pyrimidine dimers (CPDs) were detected after 405 nm UVA laser micro-irradiation in non-photosensitized cells. The effects of UVA and γ-irradiation were also studied by silver staining of nucleolar organizer regions (AgNORs). This experimental approach revealed changes in the morphology of nucleoli after genome injury. Additionally, to precisely characterize DDR in locally induced DNA lesions, we analysed the kinetics of the 53BP1 protein involved in DDR by fluorescence recovery after photobleaching (FRAP).
- MeSH
- 53BP1 MeSH
- antigeny jaderné MeSH
- buněčné jadérko účinky záření MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- histony metabolismus MeSH
- kinetika MeSH
- luminescentní proteiny metabolismus MeSH
- mikroskopie metody MeSH
- myši MeSH
- poškození DNA * MeSH
- pyrimidinové dimery metabolismus MeSH
- ultrafialové záření * MeSH
- záření gama * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Cíl: Testovat korelaci změn subbazálního nervového plexu rohovky a stupně diabetické retinopatie u pacientů s diabetes mellitus 1. typu (DM 1). Metodika: Konfokální mikroskopií rohovky bylo vyšetřeno 38 pacientů rozdělených do tří skupin podle stupně diabetické retinopatie (DR) a 12 zdravých dobrovolníků. Byl hodnocen počet všech nervových vláken a počet hlavních vláken (t-CNFD, CNFD), celková délka (CNFL) a jejich tortuozita (CNFT). Výsledky: CNFD byla u pacientů bez diabetické retinopatie, s lehkým a těžkým stupněm DR nižší než u zdravých jedinců (p < 0,0001; p = 0,004; p < 0,0001). CNFD byla nižší ve skupině s těžkým než s lehkým stupněm DR (p = 0,036). T-CNFD byla nižší ve skupině bez DR a s těžkým stupněm DR než u zdravých jedinců (p = 0,024; p < 0,0001). CNFL byla nižší ve skupině bez DR a s těžkým stupněm DR než u zdravých jedinců (p = 0,007; p < 0,0001) a u pacientů s těžkým stupněm oproti lehkému stupni DR (p = 0,028). CNFT byla vyšší ve skupině s těžkým stupněm DR oproti zdravým jedincům i skupině bez DR (p < 0,0001; p = 0,001). Závěr: Prokázali jsme postižení nervových vláken rohovky ve všech skupinách pacientů s DM 1. Změny byly výraznější u pacientů bez DR než s lehkým stupněm DR. To naznačuje, že míra postižení nervových vláken rohovky a stupeň DR neprobíhají paralelně.
Aim: The aim of the study was to test possible correlation between corneal sub-basal nerve plexus changes and the grade of diabetic retinopathy (DR). Methods: A total of 38 patients with type 1 diabetes, divided into three groups according to the grade of diabetic retinopathy, and 12 age-matched healthy subjects underwent corneal confocal microscopy. Corneal main nerve fiber density (CNFD), total corneal nerve fiber density (t-CNFD), nerve fiber length (CNFL) and nerve tortuosity (CNFT) were evaluated. Results: CNFD was lower in patients without DR, with mild grade DR, and with advanced grade DR than in healthy subjects (p < 0.0001, p = 0.004 and p < 0.0001, respectively). There was also lower CNFD in patients with advanced grade DR than with mild grade DR (p = 0.036). T-CNFD was lower in patients without DR and advanced grade DR than in healthy subjects (p = 0.024 and p < 0.0001, respectively). CNFL was lower in patients without DR and advanced grade DR than in healthy subjects (p = 0.028). CNFT was higher in patients with advanced grade DR than in healthy subjects and patients without DR (p < 0.0001; p = 0.001). Conclusion: We demonstrated changes in corneal sub-basal nerve fiber count in all diabetic patient groups, with or without DR. The changes were more pronounced in patients without DR than with mild grade DR. The study suggests no direct correlation between progression of corneal nerve fiber changes and changes in DR.
- MeSH
- diabetes mellitus 1. typu komplikace MeSH
- diabetická retinopatie diagnostické zobrazování MeSH
- dospělí MeSH
- klinická studie jako téma MeSH
- konfokální mikroskopie * metody využití MeSH
- lidé středního věku MeSH
- lidé MeSH
- neuropatie tenkých vláken * diagnostické zobrazování MeSH
- rohovka * diagnostické zobrazování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Studies on fixed samples or genome-wide analyses of nuclear processes are useful for generating snapshots of a cell population at a particular time point. However, these experimental approaches do not provide information at the single-cell level. Genome-wide studies cannot assess variability between individual cells that are cultured in vitro or originate from different pathological stages. Immunohistochemistry and immunofluorescence are fundamental experimental approaches in clinical laboratories and are also widely used in basic research. However, the fixation procedure may generate artifacts and prevents monitoring of the dynamics of nuclear processes. Therefore, live-cell imaging is critical for studying the kinetics of basic nuclear events, such as DNA replication, transcription, splicing, and DNA repair. This review is focused on the advanced microscopy analyses of the cells, with a particular focus on live cells. We note some methodological innovations and new options for microscope systems that can also be used to study tissue sections. Cornerstone methods for the biophysical research of living cells, such as fluorescence recovery after photobleaching and fluorescence resonance energy transfer, are also discussed, as are studies on the effects of radiation at the individual cellular level.
The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.
