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Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (l o) phase to liquid-disordered (l d) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable. Image deconvolution and the position of the region of interest enables clear differentiation of the membrane order in the plasma membrane and intracellular membranes. [Display omitted] • Deconvolution enables membrane differentiation and higher precision GP-analyses. • The order difference of plasma and intracellular membranes is higher than recognised. • Optimising SN allows detection of more subtle GP-changes than previously possible. • Plasma membrane GP-values are best assessed slightly outside the cell. • Image deconvolution should become a routine step in GP-analyses. [ABSTRACT FROM AUTHOR]