Accessing to cellular innards water dipolar relaxation by dimethylamino-naphthalene probes and phasor analysis of multiplexing fluorescence microscopy
Belén Torrado1, Alexander Vallmitjana2, Leonel S. Malacrida3
1- Biomedical Engineering, University of California Irvine, Irvine, CA, USA. 2-Biomedical Engineering, University of California Irvine, Irvine, CA, USA. 3- Advanced Bioimaging Unit, Hospital de Clínicas and Institut Pasteur of Montevideo, Montevideo, Uruguay
Water dynamics in different subcellular locations are crucial to modulate cellular processes, such as enzyme function, membrane dynamics, and phase separation. The existence of methods to study water dynamics at the subcellular level in vivo are scarce and technically challenging. The environmentally sensitive fluorescent probes derived from dimethylaminonaphthalene (DAN) were designed by Gregorio Weber to sense polarity and dipolar relaxation around its proximity and address macromolecules' structural dynamics. In this work, we used four different DAN probes: LAURDAN, PRODAN, ADCAN, and DANCA, with a differential affinity for different subcellular locations due to a functional group included in its carbonyl group. These probes evaluate the water dipolar relaxation. Thus, they can be used to address water dynamics in vivo. Cells were stained with DAN probes and imaged using time and hyperspectral resolved microscopy using 2-photon excitation. We combined lifetime and spectral information in multidimensional analysis using the phasor approach to understand and quantify the subcellular water dynamics. This approach in combination with 6-dimensional phasor variables analysis (lifetime channel blue and green and spectral) enables us to trace the water dipolar relaxation at the cellular innards. Also, I will cover the latest developments at the lab to study the water relaxation at membrane-bound specific organelle by phasor multi-harmonic analysis of LAURDAN fluorescence with hyperspectral imaging.
To finalize, I will tribute Luis A. Bagatolli in recognition of his contribution to science and his influence on my work and friendship.