Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor

Fluorescence microscopic approaches represent powerful techniques to monitor molecular interactions in the cellular milieu. Measurements of fluorescence lifetime and anisotropy enjoy considerable popularity in this context. These measurements are often performed on live as well as fixed cells. We report here that formaldehyde-induced cell fixation introduces heterogeneities in the fluorescence emission of serotonin1A receptors tagged to enhanced yellow fluorescent protein, and alters fluorescence lifetime and anisotropy significantly. To the best of our knowledge, our results constitute the first report on the effect of formaldehyde fixation on fluorescence parameters of cellular proteins. We conclude that fluorescence parameters derived from fixed cells should be interpreted with caution.

Research highlights
► Formaldehyde-induced cell fixation introduces heterogeneities in fluorescence emission.
► Fixation significantly alters fluorescence lifetime and anisotropy of EYFP tagged serotonin1A receptor.
► Fluorescence parameters derived from fixed cells therefore must be interpreted with caution.