Measuring fast stochastic displacements of bio-membranes with dynamic optical displacement spectroscopy.

C Monzel,A-S Smith,U Seifert,K Sengupta,R Merkel,D Kirchenbüchler,D Schmidt,C Kleusch

doi:10.1038/ncomms9162

Abstract

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them. Here we introduce a novel technique—dynamic optical displacement spectroscopy (DODS), to measure stochastic displacements of membranes with unprecedented combined spatiotemporal resolution of 20 nm and 10 μs. The technique was validated by measuring bending fluctuations of model membranes. DODS was then used to explore the fluctuations in human red blood cells, which showed an ATP-induced enhancement of non-Gaussian behaviour. Plasma membrane fluctuations of human macrophages were quantified to this accuracy for the first time. Stimulation with a cytokine enhanced non-Gaussian contributions to these fluctuations. Simplicity of implementation, and high accuracy make DODS a promising tool for comprehensive understanding of stochastic membrane processes.

Highlights

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them
We introduce a novel methodology called dynamic optical displacement spectroscopy (DODS) which circumvents these issues and can measure membrane fluctuations even in the complex optical environment of nucleated cells
To perform DODS, the membrane under study is labelled with a fluorescent dye and placed in the illuminated confocal volume of a standard fluorescence correlation spectroscopy (FCS) set-up

Summary

Introduction

Stochastic displacements or fluctuations of biological membranes are increasingly recognized as an important aspect of many physiological processes, but hitherto their precise quantification in living cells was limited due to a lack of tools to accurately record them. Several techniques have been developed to measure membrane fluctuations, including flicker spectroscopy[23], contour analysis[19], diffraction phase microscopy[25] and reflection interference contrast microscopy (RICM)[20,30] These techniques often use camera based detection[17,18,19,20,21,22,23,25,29] with limited time resolution, and/or rely on refractive index induced contrast[17,18,19,20,21,22,23,24,25,28,29], which is impossible to accurately quantify in nucleated cells due to the presence of organelles and inhomogeneous protein distribution causing ill-defined variations in refractivity. In a given technique, only a specific part of the cell could be accessed—for example, along the equator[23,25] or close to a substrate[1,3,28]

Methods

Results

Conclusion