Abstract
Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel of the image is associated with a phasor and each phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of LAURDAN in each pixel of an image.
Highlights
Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location
The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location
The biological membrane is not as simple as the cuvette sample, but we should have linear combinations independently of the complexity of the system and we can make some general predictions about the effect of polarity and dipolar relaxations if we can compare spectral and lifetime behavior at each pixel in the biological samples as we did for this example using just 3 pixels
Summary
Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. With the advances in confocal and camera based microscopy in the last two decades, lifetime and spectral information are becoming common tools to study in vivo complex photophysical processes with high spatial and temporal resolution The complementarity between these two observations, if measured together, could provide a deeper description of molecular interactions, metabolic profiles and membrane organization, among other properties[1, 2]. Fereidouni et al used the phasor approach for resolving fluorescence spectral components, to unmix fluorescence signals from multiple dye staining and to interpret the data of FRET experiments without previous knowledge about the system under study[8,9,10] Since these developments, phasor plots were applied in the biological field to analyze complex fluorescence processes[11,12,13,14,15,16]. Examples of this approach are the spectral resolved fluorescence lifetime microscope with a multi-anode PMT, where seven[20], sixteen[17] or thirty-two[18] parallel channels are used
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
