Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy

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.

Dynamic Excimer (DYNEX) Imaging of Lipid Droplets

“…Next, we employed DYNEX imaging, combined with a validated multiparametric microscopy approach for determining microenvironment polarity, to compare the polarity inside lipid droplets in the human colon cell line CCD-18Co and the mouse colon carcinoma cell line CT26.WT. The polarity in lipid droplets is an indirect estimation of the lipid content and composition and can be related to altered metabolic states .…”

“…The polarity in lipid droplets is an indirect estimation of the lipid content and composition and can be related to altered metabolic states . For these measurements, once lipid droplet pixels were segmented using DYNEX, we extracted polarity E T 30 values (Figure ) via multiparametric microscopy by combining the time-gated intensity ratio and fluorescence lifetime information (see the SI for experimental details and Figures S11–S13). Heretofore, there have been some efforts to characterize intracellular polarity employing fluorescence microscopy techniques using emission spectra, fluorescence intensity, and ratiometric measures. − Nevertheless, biological samples present important difficulties for quantitative imaging that are overcome by our methodology …”

“…In a previous work, we designed a robust and quantitative methodology to obtain intracellular polarity in terms of the E T 30 parameter by multiparametric microscopy and simultaneously employing two orthogonal imaging approaches, namely, time-gated ratiometric and fluorescence lifetime imaging . The E T 30 parameter, defined as the difference in energy between the excited state and the ground state of the pyridinium- N -phenolate of betaine, has an advantage over other polarity parameters combining general solvent effects and hydrogen-bonding interactions .…”

“…Taking advantage of this property, herein, we propose a new approach to organelle-specific tracking using a new layer of information based on the kinetics of the acridone excited state. This segmentation approach is subsequently combined with a previously validated method to probe polarity microenvironments, resulting in a novel imaging approach. The novelty of this DYNamic EXcimer (DYNEX) imaging method relies on the sensitive and specific detection of the dynamic excited-state formation of excimers of acridone 1 inside live cells.…”

Chimeric Drug Design with a Noncharged Carrier for Mitochondrial Delivery

“…Acridone derivatives are known to be very good DNA intercalating agents and therefore usually accumulate in the nucleus [ 41 ]. They are also known to have very long fluorescence lifetimes, τ, which make them very suitable for FLIM imaging [ 46 , 47 , 48 ]. In fact, probe 1 permits the quantitative determination of microenvironment dipolarity inside mitochondria using the excellent sensing capabilities of the acridone moiety [ 40 ].…”