Bipolar and fixable probe targeting mitochondria to trace local depolarization via two-photon fluorescence lifetime imaging

Abstract

Polarization/depolarization levels of different single mitochondria in a cell are inhomogeneous, and always varying. Because depolarization is an indicator of mitochondrial dysfunction, tracing local depolarization is highly desirable. The existing fluorescent probes, however, are not well suited for this task, although they are applicable to assess the average polarization extents of whole cells. A multifunctional and bipolar probe MITFPS is thus developed, which includes a positively charged hydrophilic group and an environment sensitive fluorophore. In the probe design, the hydrophilic anchoring unit is chemically immobilized on a membrane protein, while the lipophilic fluorophore can be inserted deep into the phospholipid layer. The probe exhibits a sensitive response to the local variation in polarization by changing its fluorescence lifetime. MITFPS's applicability is confirmed by real-time in situ imaging of the complete process of an uncoupler-induced depolarization under a two-photon fluorescence lifetime microscope. The imaging result reveals that one mitochondrion could have quite different polarization than the other, even though they are in the same cell.

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

“…Measuring subcellular activity in living cells with fluorescent sensors is a powerful means of collecting information specific to a particular subcellular microenvironment . In particular, mitochondria, the powerhouses of the cell, possess a variety of physicochemical properties such as membrane potential, micropolarity, local microviscosity, proton gradient, ionic transport, redox environment, and temperature that are of interest owing to their influence on the efficiency of mitochondrial metabolism . Mitochondrial micropolarity ( ϵ m ), mitochondrial membrane potential (Δ ψ m ), and local microviscosity ( η m ) are particularly important as they affect cellular bioenergetics, ATP production, reactive oxygen species (ROS) production and regulation, and the initiation of cell‐death pathways.…”

Responsive hetero-organelle partition conferred fluorogenic sensing of mitochondrial depolarization

“…Recently, detection of mitochondrial depolarization was achieved using a bipolar probe featuring a positively charged hydrophilic group and an environment sensitive fluorophore which exhibits an altered fluorescence lifetime related to membrane polarization. 5 Herein we report responsive hetero-organelle partition mediated detection of mitochondrial depolarization using RC-TPP, which preferentially accumulates in mitochondria to give blue fluorescence. RC-TPP relocates from depolarized mitochondria into acidic lysosomes to give intense rhodamine fluorescence, allowing a signal-on report of mitochondrial depolarization (Fig.…”

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

“…[1][2][3][4][5][6][7][8][9] In particular,mitochondria, the powerhouses of the cell, possess av ariety of physicochemical properties such as membrane potential, micropolarity,local microviscosity,proton gradient, ionic transport, redox environment, and temperature that are of interest owing to their influence on the efficiency of mitochondrial metabolism. [3,[10][11][12][13][14][15][16][17][18][19][20] Mitochondrial micropolarity (e m ), mitochondrial membrane potential (Dy m ), and local microviscosity (h m )a re particularly important as they affect cellular bioenergetics,A TP production, reactive oxygen species (ROS) production and regulation, and the initiation of cell-death pathways.…”