The Laurdan Spectral Phasor Method to Explore Membrane Micro-heterogeneity and Lipid Domains in Live Cells

Abstract

In this method paper we describe the spectral phasor analysis applied to Laurdan emission for the assessment of the fluidity of different membranes in live cells. We first introduce the general context and then we show how to obtain the spectral phasor from data acquired using a commercial microscope.

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

“…After the analysis we found two interesting features. First, in agreement with previous observations [2,40], cell membranes other than LB-like structures (green cursor, Figure 5A and B) fall between the limits of the l d − l o trajectory defined by the lamellar model membranes used as references (red and purple cursors respectively, Figure 5A and B). This observation indicates that the physical features of these membranes can be described as a linear combination of the l d and l o phases as observed in our reference membrane models.…”

“…The fluorescence spectra at each pixel from the LAURDAN fluorescence spectral images were Fourier transformed, as previously described [1,2]. Briefly, the LAURDAN spectrum obtained for each pixel is transformed using the following mathematical expressions: $$\mathrm{normalx}\text{ coordinate}=G=\frac{{\mathrm{\Sigma }}_{\lambda }Ifalse(\lambda false).\text{italiccos}false(2\pi nfalse(\lambda -\lambda ifalse)/Lfalse)}{{\mathrm{\Sigma }}_{\lambda }Ifalse(\lambda false)}$$ $$\mathrm{normaly}\text{ coordinate}=S=\frac{{\mathrm{\Sigma }}_{\lambda }Ifalse(\lambda false).\text{italicsin}false(2\pi nfalse(\lambda -\lambda ifalse)/Lfalse)}{{\mathrm{\Sigma }}_{\lambda }Ifalse(\lambda false)}$$ where I(λ) is the intensity at each channel step of the spectrum (32 steps), n is the number of the harmonic and L the length of the spectrum taken (728–416 = 312 nm for our experiments).…”

“…The spectral phasor analysis appeared a few years ago as a useful method to unmix fluorescence spectral images obtained from multiple-labeled specimens with minimal input [1] or to analyze the complex fluorescence emission of the well-known LAURDAN properties to study membrane dynamics [2]. This type of analysis has emerged as a promising tool to disentangle relevant aspects of distinct membranous systems.…”

“…different methods are available to perform non-invasive studies of living specimens at different length and time scales. As mentioned above, spatially resolved spectral data obtained using the fluorescent membrane probe 6-dodecanoyl-2-dimethylamino (LAURDAN) can be analyzed by the spectral phasor method [2]. This method is based on the Fourier transformation of the emission spectra of LAURDAN (that can be also used to analyze spectral information obtained from a fluorimeter [28]) and have been shown to be a powerful tool to analyze data from cellular and model membranes [2,28].…”

“…As mentioned above, spatially resolved spectral data obtained using the fluorescent membrane probe 6-dodecanoyl-2-dimethylamino (LAURDAN) can be analyzed by the spectral phasor method [2]. This method is based on the Fourier transformation of the emission spectra of LAURDAN (that can be also used to analyze spectral information obtained from a fluorimeter [28]) and have been shown to be a powerful tool to analyze data from cellular and model membranes [2,28]. These Fourier transformations allow one to determine, with a simple visual inspection, the presence of complex interactions in the LAURDAN/membrane system without the assumption of a particular model as required by the classical Generalized Polarization function [28].…”