Abstract
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.
Highlights
We describe a recently developed, accurate method for sensing intracellular polarity, using multiparametric fluorescence microscopy
Sensing microenvironment polarity is of especial interest for intracellular studies, since many different cellular processes, such as recognition interactions and biochemical reaction kinetics that include metabolism, membrane transport, immune response, degradation of cellular components or cellular aging, among others, are controlled by the general and specific features of the environment [1]
Our method overcomes the limitations of conventional ratiometric approaches by taking advantage of multidimensional fluorescence conventional taking advantage of multidimensional fluorescence microscopy microscopy ratiometric to performapproaches a reliablebyand robust multilinear calibration that allows quantitative to perform a reliable and robust multilinear calibration that allows quantitative determination of determination of intracellular polarity
Summary
We describe a recently developed, accurate method for sensing intracellular polarity (dipolarity), using multiparametric fluorescence microscopy. Several methods to study intracellular polarity, based on luminescent probes, have been reported. Most of these methods are based on ratiometric sensors, using dual-channel fluorescence microscopy. We present a new methodology aimed at exploring the local polarity of heterogeneous media, and we applied it for cell imaging in order to obtain an intracellular polarity map [12]. For this purpose, we employ the photophysical information provided by a fluorophore that is extremely sensitive to the local polarity environment.
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