Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools

Abstract

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.

Dynamics of transition dipole moment orientation in representative fluorescent proteins

“…Our results largely explain a seeming contradiction between the published virtually identical mean xTDM and mTDM orientations in the studied FPs, 8 and observations of time-resolved fluorescence anisotropies that suggest differences between xTDM and mTDM orientations within the FP molecular framework. The results will allow better understanding of widths of orientational distributions observed in model membranetethered FP-bearing constructs, 29 as well as in other FP-labeled proteins, such as G proteins, 30,31 in which the molecular orientation of the FP moiety reports on the functional state of the studied protein. By allowing more accurate insights into the structure of FP-bearing membrane proteins, our results will aid in rational development of genetically encoded probes of G protein signaling 32 and other cellular processes, as well as in elucidating molecular mechanisms of cell signaling.…”

Novel Genetically Encoded Biosensors for Functional Imaging of Cell Signaling by Polarization Microscopy

“…Cells expressing constructs with mScarlet and mCherry were imaged using 543 nm excitation; emission 560-650 nm was collected. The resulting images were processed and analyzed as described [42], using Fiji and publicly available macros for LD image analysis, as well as Polaris+ software (Innovative Bioimaging, L.L.C. ).…”

“…Agonists and antagonists were purchased from Sigma-Aldrich. Microscopy LD imaging was carried out as described previously 8,42 , on a customized laser scanning confocal microscope (Olympus FV1200), equipped with a polarization modulator (RPM-2P, Innovative Bioimaging, L.L.C.) alternating the excitation light linear polarization between acquisition of subsequent pixels by the microscope.…”

A genetically-encoded nanobody sensor reveals conformational diversity in β-arrestins orchestrated by distinct seven transmembrane receptors

“…Single-photon polarization microscopy was performed as described previously 34,54 using an Olympus FV1200 confocal microscope equipped with a polarization modulator (RPM-2P, Innovative Bioimaging) alternating the direction of the excitation light polarization between acquisition of subsequent pixels. Ib32-meGFP-H-Ras was observed using laser light of 488 nm wavelength and 80 µW intensity, through a 40X water immersion objective lens (UApoN340, NA1.15, Olympus, Japan).…”