The spectral phasor approach to resolving membrane order with environmentally sensitive dyes

Area of Science:

• Biophysics
• Spectroscopy
• Microscopy

Background:

• Hyperspectral imaging captures 3D spectral data for precise sample characterization.
• Confocal microscopes increasingly offer hyperspectral imaging capabilities.
• Spatially resolved spectroscopic data is crucial for understanding complex samples.

Purpose of the Study:

• To apply spectral phasor analysis to hyperspectral images for detailed membrane analysis.
• To investigate the utility of environmentally sensitive dyes with phasor analysis for membrane heterogeneity.
• To resolve lipid packing and hydration changes in model membranes using LAURDAN probe.

Main Methods:

• Acquisition of hyperspectral images using confocal microscopy.
• Application of spectral phasor analysis to transform pixel-wise spectra into a polar plot.
• Utilizing the LAURDAN probe on model membranes to probe membrane properties.

Main Results:

• Spectral phasor analysis provides quantitative and graphical insights into fluorophore spectral characteristics.
• The combination of hyperspectral imaging and phasor analysis effectively measures lateral membrane heterogeneity.
• Distinct spectral features related to packing and hydration were resolved in model membranes.

Conclusions:

• Spectral phasor analysis of hyperspectral images is a powerful tool for studying membrane heterogeneity.
• The method is effective for resolving small changes in lipid packing and hydration.
• This approach has broad applicability to various dyes and complex biological systems, including cell membranes.