Defining the subcellular interface of nanoparticles by live-cell imaging

• Published in: PloS one Vol. 8; no. 4; p. e62018
• Main Authors: Hemmerich, Peter H, von Mikecz, Anna H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26.04.2013; Public Library of Science (PLoS)
• Subjects: Active transport; Animals; Biology; Biophysics; Cell Survival; Cells (biology); Chemistry; Confocal microscopy; Correlation; Cytoplasm; Diffusion; Dynamin; Endocytosis; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence spectroscopy; Gene expression; HeLa Cells; Humans; Hydrazones - metabolism; Imaging techniques; Intracellular; Materials Science; Mice; Microenvironments; Microscopy; Mitochondria; Morphology; Nanomaterials; Nanoparticles; Nanoparticles - chemistry; Nanoparticles - ultrastructure; Nanotechnology; NIH 3T3 Cells; Nuclear reactions; Nuclear transport; Optical Imaging - methods; Organelles; Particle motion; Particle Size; Photobleaching; Polystyrene; Polystyrene resins; Polystyrenes - metabolism; Proteins; Quantum dots; Recovery (Medical); Silica; Silicon dioxide; Silicon Dioxide - chemistry; Spectroscopy; Subcellular Fractions - metabolism; Surface chemistry; Translocation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0062018

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.