Definition of phosphoinositide distribution in the nanoscale

New functionalities of phosphoinositides (PIs) are being revealed continuously, and the scale of the membrane area studied is becoming smaller, from the micrometer range like the entire surface of organelles to the nanometer range as in subdomains of organelles. Concurrently, function of less abundant PIs, such as PI(3,4)P₂ and PI(3,5)P₂, attracts increasing attention. In accordance with the progress, finer and more accurate information on PI distribution is required. The fluorescence biosensor method utilizing PI-binding domains and/or immunolabeling with anti-PI antibodies are used for this purpose in most studies but both methods are known to have caveats. In this article, we examined how PI distribution was defined in recent studies and discussed whether methodological uncertainty has any bearing on the results.