A method for high-throughput measurements of viscosity in sub-micrometer-sized membrane systems

Chwastek, G.; Petrov, E. P.; Sáenz, J. P. (2019) ChemBioChem, 21(6), 836–844. DOI: 10.1002/cbic.201900510

Summary

Studying membrane adaptation in small systems like bacterial cells requires robust, high-throughput approaches to characterize membrane fluidity. We developed a fast, financially accessible method to measure membrane microviscosity in bulk suspension using a standard plate reader. The approach exploits the viscosity-sensitive fluorescence of DCVJ (9-(2,2-dicyanovinyl)julolidine), a twisted intramolecular charge transfer (TICT) dye whose brightness follows a power-law dependence on viscosity. We validated the method using artificial membranes across a range of compositions and temperatures, achieving good agreement with published values. As a proof of concept, we detected a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.

Key Findings

• DCVJ fluorescence brightness (R = integrated emission / absorbance) follows a power-law relationship with viscosity, enabling quantitative viscosity measurement from a simple plate reader.
• Viscosity values and activation energies for model lipid membranes (DOPC, DPPC, sphingomyelin, cholesterol mixtures) agree with published FCS and FRAP measurements.
• A lipid phase transition was detected in Mycoplasma mycoides membranes, demonstrating the method’s applicability to living cells.
• The approach overcomes limitations of existing methods: FCS requires specialized microscopy, FRAP cannot resolve sub-micrometer vesicles, and fluorescence lifetime imaging is expensive and low-throughput.
• The dual measurement of absorbance and fluorescence corrects for variations in dye incorporation, improving accuracy across samples.

Methods

• DCVJ dye: incorporated into liposomes or cell membranes at sub-mol% concentrations.
• Plate reader assay: simultaneous measurement of absorbance (to quantify dye incorporation) and fluorescence emission (viscosity-dependent) on a standard 96-well plate reader.
• Calibration: DCVJ brightness measured in glycerol and ethylene glycol at varying temperatures to establish the power-law relationship.
• Validation: liposomes of DOPC, DPPC, SM, and SM/cholesterol mixtures measured across 10–60 °C.
• Biological application: M. mycoides membranes measured at varying temperatures.

Significance

This paper provided a practical tool for the lab’s membrane research program. High-throughput viscosity measurement enabled the screening of bacterial strains, mutants, and lipid conditions that would later become central to the minimal cell and lipidome design work. The detection of a phase transition in M. mycoides foreshadowed the later discovery that this organism’s lipidome can be tuned and minimized.