Applying first & second harmonic spectral phasor analysis on a single-wavelength calcium fluorophore

Ratiometric and single-wavelength fluorophores are limited in their ability to provide basal level activity prior to treatment. This study presents a novel approach to characterise cell-wide basal activity of calcium using first and second harmonic spectral phasor analysis. Cells stained with the single-wavelength calcium fluorophore Oregon Green™ BAPTA 1-AM exhibited significant differences in wavelength or width in the nucleus, cytoplasm and membrane under basal conditions. Large and small cursor analysis was applied in the first and second harmonic, with smaller cursors revealing a region of interest enveloping and protruding from the nucleus in a structure akin to the sarcoplasmic reticulum. The first harmonic was found to be more sensitive in measurements of <i>λ_max</i> , while the second harmonic showed increased sensitivity in measurements of spectral width. The results of this study indicate that first and second harmonic frequencies should be used in conjunction with phasor analysis of fluorophore microenvironments, rather than the first harmonic alone. Use of this approach may provide more insight into the cellular microenvironment under basal activity and treatment responses.