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

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.

• MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• konformace proteinů MeSH
• membránové proteiny metabolismus   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• polarizační mikroskopie metody   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH