3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics

Miguel Sison,Theo Lasser,Amir Nahas,Tanja Weil,Sabyasachi Chakrabortty,Jérôme Extermann,Paul James Marchand,Antonio Lopez

doi:10.1038/srep43275

Abstract

We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume.

Highlights

Confocal fluorescence microscopy remains the most widely used technique for cellular imaging
According to the MALDI-ToF mass spectra (Fig. 1b), about 19 triphenyl phosphonium groups (TPP) groups were attached to the Cationized bovine serum albumin (cBSA) surface yielding cBSA-TPP in good yields. cBSA-TPP was purified and subjected to AuNP preparation
Since the stability of the cBSA-TPP passivated AuNPs in biological media is crucial for cellular uptake and subsequent observation of their movement without prior aggregation, dynamic light scattering experiments were performed in DMEM medium

Summary

Introduction

Confocal fluorescence microscopy remains the most widely used technique for cellular imaging. 0 400 500 600 700 Wavelength (nm) at the particle surface after coating[12] In this way, photothermal imaging AuNPs could be envisaged carrying the desired functionalities for targeting sub-cellular organelles, such as mitochondria, inside living cells. The photothermal contrast mechanism relies on a temporally modulated refractive index in the near vicinity of the AuNPs resulting from heat dissipated by the AuNPs’ plasmon-enhanced absorption This contrast mechanism is the basis for photothermal optical lock-in optical coherence microscopy (poli-OCM), which provides two distinct imaging modalities: a dark-field mode (dfOCM) for imaging the 3D cell volume, and a poli-mode, utilizing functionalized AuNPs for highly specific 3D mitochondria imaging[13]

Methods

Results

Conclusion