Nejvíce citovaný článek - PubMed ID 19211795
Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.
- MeSH
- buněčná membrána metabolismus MeSH
- nanotechnologie * MeSH
- receptory buněčného povrchu * metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- receptory buněčného povrchu * MeSH
Fresnel incoherent correlation holography (FINCH) was a milestone in incoherent holography. In this roadmap, two pathways, namely the development of FINCH and applications of FINCH explored by many prominent research groups, are discussed. The current state-of-the-art FINCH technology, challenges, and future perspectives of FINCH technology as recognized by a diverse group of researchers contributing to different facets of research in FINCH have been presented.
- Klíčová slova
- Fresnel incoherent correlation holography, color holography, computational coherent superposition, digital holographic microscopy, digital holography, fluorescence microscopy, incoherent holography, lattice light-sheet holography, metasurfaces, multiplexed imaging, phase-shifting interferometry, single-molecule localization microscopy,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.
- Klíčová slova
- cell nucleus, gene expression, photoactivation, stimulated emission depletion, stochastic optical reconstruction, structured illumination, super-resolution microscopy, transcription factors, transcription foci,
- MeSH
- fluorescenční mikroskopie * metody MeSH
- genetická transkripce * MeSH
- lidé MeSH
- regulace genové exprese * MeSH
- RNA-polymerasa II metabolismus MeSH
- transkripční faktory metabolismus MeSH
- vazba proteinů MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- RNA-polymerasa II MeSH
- transkripční faktory 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
With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.
