Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

• MeSH
• analýza jednotlivých buněk * MeSH
• fluorescenční barviva metabolismus   MeSH
• fluorescenční mikroskopie * MeSH
• HEK293 buňky MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• navrhování softwaru * MeSH
• počítačové zpracování obrazu * MeSH
• polarizační mikroskopie * MeSH
• proteiny vázající GTP genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• signální transdukce MeSH
• simulace molekulární dynamiky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva MeSH
• luminescentní proteiny MeSH
• proteiny vázající GTP MeSH
• rekombinantní fúzní proteiny MeSH

For decades, biologists have relied on software to visualize and interpret imaging data. As techniques for acquiring images increase in complexity, resulting in larger multidimensional datasets, imaging software must adapt. ImageJ is an open-source image analysis software platform that has aided researchers with a variety of image analysis applications, driven mainly by engaged and collaborative user and developer communities. The close collaboration between programmers and users has resulted in adaptations to accommodate new challenges in image analysis that address the needs of ImageJ's diverse user base. ImageJ consists of many components, some relevant primarily for developers and a vast collection of user-centric plugins. It is available in many forms, including the widely used Fiji distribution. We refer to this entire ImageJ codebase and community as the ImageJ ecosystem. Here we review the core features of this ecosystem and highlight how ImageJ has responded to imaging technology advancements with new plugins and tools in recent years. These plugins and tools have been developed to address user needs in several areas such as visualization, segmentation, and tracking of biological entities in large, complex datasets. Moreover, new capabilities for deep learning are being added to ImageJ, reflecting a shift in the bioimage analysis community towards exploiting artificial intelligence. These new tools have been facilitated by profound architectural changes to the ImageJ core brought about by the ImageJ2 project. Therefore, we also discuss the contributions of ImageJ2 to enhancing multidimensional image processing and interoperability in the ImageJ ecosystem.

• Klíčová slova
• Fiji, ImageJ, image analysis, imaging, microscopy, open source software,
• MeSH
• počítačové zpracování obrazu * MeSH
• software * MeSH
• umělá inteligence * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications.

• Klíčová slova
• fluorescent protein, polarization microscopy, transition dipole moment,
• MeSH
• anizotropie MeSH
• červený fluorescenční protein MeSH
• krystalografie rentgenová MeSH
• luminescentní proteiny chemie   genetika   MeSH
• polarizační mikroskopie MeSH
• světlo MeSH
• zelené fluorescenční proteiny chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luminescentní proteiny MeSH
• zelené fluorescenční proteiny MeSH