Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential

Dietrich W M Walsh,Philippe Barberet,Hervé Seznec,Gabriele Multhoff,Stefanie Girst,Giovanna Muggiolu,Christian Siebenwirth,Christoph Greubel,Judith Reindl,Thomas E Schmid,Katarina Ilicic,Guenther Dollinger,Marina Simon,Hans Zischka

doi:10.1038/srep46684

Abstract

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 μm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria’s response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

Highlights

IntroductionDue to the nature of energy deposition of lasers, such experiments do not enable a quantification of the energy deposited in individual mitochondria and only a minor fraction of molecular species may be affected
Polarized mitochondria labelled with Tetramethyl rhodamine ethyl ester (TMRE) were visualized in both A549 and MCF7 cell lines and irradiated with either arrays of irradiation points targeted to single (Fig. 1a) or clusters of mitochondria (Fig. 1b) using both 55 MeV carbon ions (SNAKE) or 3 MeV protons (AIFIRA)
To deposit the same amount of energy as 1 carbon ion applied at SNAKE, 35 protons were used at AIFIRA

Summary

Introduction

Due to the nature of energy deposition of lasers, such experiments do not enable a quantification of the energy deposited in individual mitochondria and only a minor fraction of molecular species may be affected This is where particle radiation and ion beams become invaluable radiation techniques. Tetramethyl rhodamine ethyl ester (TMRE) a cationic fluorophore, which accumulates electrophoretically in polarized mitochondria[14] enables the assessment of mitochondrial membrane potential, and mitochondrial function, allowing for changes in membrane potential to be visualized very rapidly[15]. This membrane permeable dye allows for a simple “on/off ” readout of fluorescent signal accumulation in mitochondria in direct relation to the mitochondrial membrane potential ΔΨm. The techniques enable the exploration of the response of mitochondria to highly specific energy deposition with beamspot sizes which are in the size range of the mitochondria themselves

Methods

Results

Conclusion