Nejvíce citovaný článek - PubMed ID 22002026
Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). FINDINGS: Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. CONCLUSIONS: Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.
- MeSH
- algoritmy MeSH
- Bayesova věta MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- fluorescenční mikroskopie MeSH
- králíci MeSH
- lidé MeSH
- počítačové zpracování obrazu metody MeSH
- software MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Advanced bioimaging uncovers insights into subcellular structures of plants.
Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.
- MeSH
- Arabidopsis MeSH
- intravitální mikroskopie metody MeSH
- rostlinné buňky * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.
- MeSH
- Arabidopsis metabolismus MeSH
- epidermis rostlin cytologie metabolismus MeSH
- hypokotyl cytologie metabolismus MeSH
- konfokální mikroskopie MeSH
- mikroskopie metody MeSH
- mikrotubuly metabolismus MeSH
- mutace genetika MeSH
- osvětlení * MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny huseníčku MeSH
- rekombinantní fúzní proteiny MeSH
- zelené fluorescenční proteiny MeSH
