LAURDAN fluorescence and phasor plots reveal the effects of a H2O2 bolus in NIH-3T3 fibroblast membranes dynamics and hydration

Abstract

Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effects of an HO bolus on NIH-3T3 membranes dynamics monitored by LAURDAN fluorescence. Exposure of NIH-3T3 cells to a bolus of HO modifies the cell membranes and, in particular, the plasma membrane in a complex manner. The LAURDAN results reveal that the peroxide treatment decreases membrane fluidity but surprisingly increases dipolar relaxation around the excited probe. Using the Multidimensional-phasor approach we elucidated the complex photophysics of LAURDAN incorporated into cell membrane after HO exposure. The results indicate the occurrence of LAURDAN fast-diffusion from gel↔l phases in membranes exposed to a HO bolus. An ad hoc hypothesis is presented to interpret the results in the context of HO oxidative distress/eustress.

Membrane Remodeling by Arc/Arg3.1

“…Thus, in contrast to HIV-Gag, the interaction of Arc with membranes is not appreciably enhanced by the presence of phosphoinositides. Hence, our initial studies of Arc-mediated effects on membrane morphology were conducted using GUVs generated from dioleoyl PC (DOPC) and labeled with LAURDAN as previously described (see, for example ( Bagatolli et al, 2003 ; Pott et al, 2008 ; Malacrida and Gratton, 2018 ; Castro-Castillo et al, 2019 )).…”

StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis

“…In the so called "blue filter," we can measure the amount of l o and l d phase in a membrane, which is affected by the amount of cholesterol (more cholesterol results in more l o phase). Cholesterol also affects the rate of dipolar relaxation in a membrane and this signal is detected in the "green filter" (more cholesterol results in an increase in dipolar relaxations) (48,49). Given the focus of this work in detecting the spatial and temporal distribution of cholesterol, we opted to use both methods to unequivocally assign the spectroscopic changes to cholesterol concentration.…”

The DIVER Microscope for Imaging in Scattering Media

“…In the spectral phasor approach the spectra are transformed to the real and imaginary components of the Fourier transformation, then the spectral shift and broadening of the full width at the half maximum (FWHM) are related to the phase increase or modulation decrease, respectively [28,29]. Unlike deconvolution, the spectral phasor approach does not require the knowledge of a spectral basis set for the analysis, which are instead needed for a classical fitting approach [29,30]. In particular, the application of spectral phasors to quantify dipolar relaxations at the interphase of membranes using LAURDAN fluorescence is important in simplifying the study of membrane dynamics and structures in cells and tissues [30,31].…”

“…Unlike deconvolution, the spectral phasor approach does not require the knowledge of a spectral basis set for the analysis, which are instead needed for a classical fitting approach [29,30]. In particular, the application of spectral phasors to quantify dipolar relaxations at the interphase of membranes using LAURDAN fluorescence is important in simplifying the study of membrane dynamics and structures in cells and tissues [30,31].…”