Single-particle Tracking as a Quantitative Microscopy-based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical Nanoparticles

Abstract

Highly sensitive fluorescence microscopy techniques allow single nanoparticles to be tracked during their uptake into living cells with high temporal and spatial resolution. From analysis of the trajectories, random motion can be discriminated from active transport and the average transport velocity and/or diffusion coefficient determined. Such an analysis provides important information regarding the uptake pathway and location of viruses and nanoparticles. In this review, we give an introduction into single-particle tracking (SPT) and determination of the mean-squared displacement. We also give an overview of recent advances in SPT. These include millisecond alternating-laser excitation for removal of spectral crosstalk, alternating wide-field (WF), and total internal reflection fluorescence (TIRF) microscopy for sensitive experiments at the plasma membrane and three-dimensional tracking strategies. Throughout the review, we highlight recent advances regarding the entry (and egress) of natural and artificial viruses obtained via SPT.

Nanoparticles With Aggregation-Induced Emission for Monitoring Long Time Cell Membrane Interactions

“…In the last decade, along with the thriving of research in the single molecule and subwavelength level, the TIRFM system has been utilized and developed extensively. TIRFM has been used in technical innovations such as smFRET [17], TIR-FCS [10], SPT [9] for single molecule detection and SIM [18], STORM [19] for fluorescence super-resolution microscopy and these reflect its flexibility in the related research. The combination of both advantages of AIE nanoparticles and the TIRFM system provides a fresh optional cell tracing tool and allows for more detailed studies of biological process in this platform.…”

“…In addition, it is predicted that if a higher concentration of the aggregates is encapsulated in by the silica, the photobleaching resistance of this single nanoparticle could be strengthened. It provides an alternative probe for the high sensitive fluorescence microscopy techniques, such as single particle tracking (SPT), which allows an improved temporal and spatial resolution for the cell-particle interaction detection [9].…”

“…It decays exponentially from the boundary of the medium of lower refractive index. Usually, the penetration depth of EW is of the order of the excitation light wavelength (50 nm ∼ 250 nm) [8,9] and varies accompanied with the angle adjustment of total reflection. Because of this thin film excitation, only the fluorophores close enough to the interface could be excited, and the out-of-focus fluorescence is suppressed to a large extent.…”

Platinum-coated Core-Shell Gold Nanorods as Multifunctional Orientation Sensors in Differential Interference Contrast Microscopy

“…[1][2][3] The majority of the SPT techniques have been focused on tracking the translational motion of a nanoobject in three-dimensional (3D) space. However, single-particle rotational tracking with orientation probes is of great importance because many chemical or biological processes also involve rotational motions at the nanoscale, including myosin walking, 4 RNA folding, 5 twisting of the dynamin assembly, 6 and self-rotation of ATPase.…”

The emergence of multiple particle tracking in intracellular trafficking of nanomedicines

“…However, the investigation of nanoparticle trafficking in complex intracellular environments has focused largely on static or bulk particle transport properties, which may not reveal individual particle interactions with their cellular environments. Following the landmark paper by Suh and coworkers (Suh et al 2003), several investigators have applied real-time multiple-particle tracking (MPT) to study the dynamic transport of nanoparticles in live cells (Bausinger et al 2006;Bergen and Pun 2008;de Bruin et al 2007;Payne 2007;Ruthardt and Brauchle 2010;Ruthardt et al 2011;Suk et al 2007). MPT is valuable in obtaining quantitative (such as diffusivity and velocity) and qualitative (such as directionality and transport mode classification) information at the individual particle level, which can be critical in revealing cellular processes that control the overall bulk transport properties.…”