Mitochondrial dynamics refers to the processes of fusion, fission, and transport that aid mitochondria in accomplishing their many roles; including ATP production, oxygen sensing, and homeostasis. Due to their involvement in numerous essential cellular activity, dysfunctional mitochondria have been implicated in a wide range of human diseases.
Confocal microscopy using fluorophores for molecular specificity remains the gold standard of intracellular imaging. However, fluorescent labels can be toxic to the cell upon prolonged exposure and still suffer from photobleaching. These compromise the application of confocal fluorescence microscopy for true long lasting time-lapse imaging of living samples.
Optical coherence microscopy (OCM) exploits the intrinsic variation in the scattering properties of the sample to achieve fast, label-free, and highly sensitive three-dimensional imaging. Unfortunately, being label-free means OCM lacks specificity and coherence based imaging techniques have no counterpart to fluorescent markers. The invention of the photothermal optical lock-in OCM (poli-OCM) brought about the possibility of specific OCM imaging using gold nanoparticles (AuNP) as photothermal bio-markers. The use of AuNPs as specific contrast agents has substantial advantages stemming from their well-established biocompatibility and photostability.
Microscopic techniques that offer fast three-dimensional imaging over extended time durations would do well in revealing previously inaccessible knowledge on mitochondria. In this work, we quantify mitochondrial dynamics based on specific poli-OCM and surface functionalization of AuNPs.
In realizing mitochondria specific poli-OCM imaging, it is necessary to functionalize AuNP with mitochondria targeting capabilities. We present copolymer surface coatings that provide the AuNPs with improved stability, solubility, and cellular uptake on top of mitochondria labeling. We further optimize the utilization of these AuNP labels for poli-OCM imaging. We also demonstrated poli-OCM imaging with differently structured gold nanolabels, which could lead to the realization of multimodal imaging using a single bio-marker.
The two quantification techniques we developed were based on (1) temporal autocorrelation analysis combined with a classical diffusion model and (2) single particle tracking. Autocorrelation analysis is the foundation of fluorescence correlation spectroscopy (FCS); a technique extensively used for analyzing dynamic phenomena in chemistry and biophysics. We extend this analysis to three-dimensional poli-OCM imaging allowing us to map quantified mitochondrial diffusion parameters in three dimensions within the cell. We also investigate how the size of the mitochondria with respect to the point spread function (PSF) of the poli-OCM impacts the result of our autocorrelation analysis. Single particle tracking complements our temporal autocorrelation analysis since recent advances in localization and tracking algorithms have demonstrated precision better than the size of the PSF.
Finally, we demonstrate the possibility of using mitochondria specific poli-OCM imaging with the quantification techniques we developed in studying the Cockayne syndrome (CS). CS is a very rare and fatal genetic disease that has been associated with mitochondrial dysfunction. To our knowledge, no study has been conducted focusing on quantifying the effect of CS on mitochondrial dynamics.