Lifetime and spectral phasors: exploiting Laurdan's fluorescence to characterize cell membranes

Cell membranes constitute the barrier between the cell interior and the external world. The composition and physical properties of cell membranes influence many cellular functions One biophysical parameter found to be especially critical is membrane fluidity; changes occurring in membrane fluidity play a key role in regulation of membrane properties under physiological conditions and in pathogenesis of disease (such as motility and metastatic potential of cancer cells). Laurdan is a fluorescent probe commonly used to image model and biological membranes. Laurdan is very sensitive to membrane packing, and therefore to membrane fluidity.;Here, we develop two novel imaging techniques to exploit Laurdan's fluorescence properties. Both techniques are based on the use of the phasor approach to analyze fluorescence images. By Fourier transformation of Laurdan lifetime decay or emission spectrum into a phasor we obtain two phasor coordinates for each pixel of an image. This approach provides a highly resolved, fast and fit-free graphical and quantitative analysis of imaging data.;First, we apply the phasor approach to fluorescence lifetime images of Laurdan in live cells. This method gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in membrane fluidity during cell migration. Then, we apply the phasor approach to Laurdan spectral images. We use this approach to perform a phasor analysis of membrane heterogeneity in NIH3T3 and HEK293 live cells. Taking advantage of the greater sensitivity to small changes of the phasor method, we are able to detect highly packed micro-domains in the cell membrane and to monitor changes in membrane packing due to acute and chronic cholesterol manipulation in live cells.