3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics

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.

High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

“…We applied our multiplane SIM system to the fast imaging of mitochondrial dynamics [18,19]. Here, SIM is an enabling imaging method, as its increase in spatial resolution and inherent background suppression allow us to visualize the mitochondria and their 3D morphology and dynamic changes thereof, which cannot be observed with conventional wide-field resolution [20].…”

High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

“…We applied our multi-plane SR-SIM system to the fast imaging of mitochondrial dynamics [18,19]. Here, SR-SIM is an enabling imaging method, as its increase in spatial resolution and inherent background suppression allows us to visualize the mitochondrial ultra-structure, the cristae, which cannot be observed with conventional wide-field resolution [20].…”

Time-Lapse Microscopy

“…TLM can also be used to study intracellular dynamics of subcellular organelles [161,162], natural cellular proteins and reporters, introduced nanoparticles and even physiological effects of small inorganic molecules and gases. Time-lapse imaging was used to monitor and quantify movements and changes in mitochondria [163][164][165]; Golgi apparatus [166]; centrosomes and microtubules [167][168][169][170]; centromeres [171]; cellular membrane [172]; dendritic spines [173]; dynamics of interkinetic nuclear migration [174,175]; intercellular uptake and distribution of nano-sized (less than 100 mkm) ceramic particles [176]; intracellular translocation of p65 and IkappaB-alpha proteins [177]; intracellular distribution of integrin beta1 and F-actin [178]; fluctuations in Notch signaling to maintain neural progenitors [179]; re-localization of PP1gamma, which is implicated in multiple cell cycle-related processes including regulation of chromosome segregation and cytokinesis [180]; movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis [181]; dynamics of 53BP1 protein in DNA-damage response [182]; measuring gene dynamics with luciferase as a reporter [183]; colocalization of MAP kinases in mitochondria [184]; clustering of acetylcholine receptor on myotubes [185]; multiple chromosomal populations of topoisomerase II [186]; focal points for chromosome condensation and decondensation [187]; intracellular calcium dynamics [188,189] and single-cell time-lapse imaging of intracellular O 2 [190].…”