Date Published: February 14, 2019
Publisher: Public Library of Science
Author(s): Ida Emilie Steinmark, Arjuna L. James, Pei-Hua Chung, Penny E. Morton, Maddy Parsons, Cécile A. Dreiss, Christian D. Lorenz, Gokhan Yahioglu, Klaus Suhling, Banafshe Larijani.
The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.
Changes in cellular viscosity have been found in a range of human diseases, including cancer , Alzheimer’s disease , Huntington’s disease , Amyotrophic lateral sclerosis , and Leigh’s syndrome , as well as during disease treatment . On a macroscopic level, viscosity is defined as the resistance to flow, but on the microscopic level—where the probe is comparable in size to the solvent molecules—it can be described, after some simplification, as the extent of free volume between solvent molecules and around the probe .
In this paper, we explored the effect of increased Ca2+ exposure on matrix viscosity and slight nutrient deprivation on membrane fluidity. In both cases, viscosity/fluidity exhibited variation, showing that these parameters are dynamic and not “fixed”, even in healthy cells—organelles may be constantly responding to environmental and physiological fluctuations. We believe this warrants further investigation because we cannot describe pathological states without first understanding the range of viscosity variation in healthy controls. It is also important to understand that, in cases where the viscosity/rigidity increases, diffusion-limited reactions will be slowed down. Conversely, higher fluidity would likely cause a rate increase. This could have potential knock-on effects for the whole organelle, cell and potentially organism. Changes in viscosity could therefore play a significant role in the mechanism behind certain illnesses; this again underlines the need to understand the physiological viscosity state. Further, it could be relevant for drug delivery systems that inherently rely on diffusion. From a methodological perspective, we have shown that a combination of chemical targeting and organelle extraction enables easy imaging of organelle viscosity and membrane fluidity. To the best of our knowledge, this is the first fully combined framework for imaging both matrix viscosity and membrane fluidity using FMR-FLIM.
Cellular viscosity imaging has so far lacked a framework for imaging both matrix/lumenal viscosity and membrane fluidity of organelles. In mitochondria, we have shown that the combination of chemical FMR targeting, organelle extraction and FMR-FLIM allow us to image the viscosity of the major regions within the organelles, namely the matrix and the membranes. We have shown that these properties are dynamic and responsive to small environmental and physiological changes, and further, we have tested the suitability of FMR-FLIM in membranes through MD simulation. We believe that single-organelle FMR-FLIM has the potential to find widespread use in biophysical, clinical and drug delivery studies.