Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model
membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the
generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral
detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane
properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets
afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly
internalized fluorescent probes.
Results: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition
to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps,
and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly
internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data
sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is
determined, which can provide insight into the mechanisms underlying changes in membrane properties and is
desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated
for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive
probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification
of the local lateral density of lipids or lipid packing.
Conclusions: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral
imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The
Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-
spectral-imaging-toolbox.