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

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