Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy González García, María del Carmen Herrero-Foncubierta, Pilar García-Fernández, Emilio Orte Gutiérrez, Ángel Biosensing Cellular microenvironment Fluorescence imaging Fluorescence Lifetime Imaging Microscopy (FLIM) Lifetime Acridones Solvatochromism M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. We thank Juan M. Cuerva and Delia Miguel (University of Granada, Spain) for the synthesis of the acridone dyes, Rafael Salto and Maria D. Giron (University of Granada, Spain) for providing the cultured cells, and Amparo Navarro and Tomas Peña-Ruiz (University of Jaen, Spain) for computational studies of the acridone dyes. M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. We thank Juan M. Cuerva and Delia Miguel (University of Granada, Spain) for the synthesis of the acridone dyes, Rafael Salto and Maria D. Giron (University of Granada, Spain) for providing the cultured cells, and Amparo Navarro and Tomas Peña-Ruiz (University of Jaen, Spain) for computational studies of the acridone dyes. The precise knowledge of intracellular polarity, a physiological parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer's. In this technical note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quantitative polarity maps. 2021-12-01T11:33:20Z 2021-12-01T11:33:20Z 2020-11-11 journal article Gonzalez-Garcia, M.C.; Herrero-Foncubierta, P.; Garcia-Fernandez, E.; Orte, A. Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy. Methods Protoc. 2020, 3, 78. [https://doi.org/10.3390/mps3040078] http://hdl.handle.net/10481/71843 10.3390/mps3040078 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España MDPI