Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Summary

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Abstract

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Introduction

The dynamics of nanoparticles (NPs) on the membrane is closely associated with the cellular uptake process, which is essential for the understanding of cell functions, viral or bacterial infections and the development of artificial nanomedical delivery systems^1,2. Single particle tracking (SPT) technique is a robust tool for characterizing the heterogeneous behaviors of NPs^3,4. In general, cell membrane is fluidic, which means that the components such as proteins and lipids can move laterally in the plasma membrane plane^5,<....

Protocol

1. Cell culture

1. Prepare complete medium for U87 MG cells by adding fetal bovine serum (final concentration 10%) and penicillin-streptomycin (final concentration 1%) to the minimum essential medium (MEM). Use plastic cell culture dish for cells subculture.
2. Passage cells 2 to 3 times a week.
   1. Remove the culture medium and rinse the cell layer with Dulbecco's phosphate-buffered saline (D-PBS) 2~3 times when confluent (80%~90%).
   2. Add 1.0 to 2.0 mL of Trypsin-EDTA solution to the cell culture dish and observe cells under an inverted microscope until cells become round (3~5 min).
   3. Add 3.0 mL of prepared complete medium an....

Results

In the protocol, the unmodified 40 x 85 nm CTAB-AuNRs were used. As shown in Figure 2B, its longitudinal plasmonic maximum at is ~650 nm (red region) and transverse resonance is at 520 nm (green region). Previous literatures have revealed that the optical properties (such as LSPR intensity) of plasmonic AuNRs will change significantly with their diameter^20,22. In Figure 2C, the scattering intensity from .......

Discussion

The presented protocol is used to study the dynamics of AuNRs on cell membrane. The protocol consists of four parts, including microscopic imaging, data extraction, dynamic parameters calculation and data analysis methods, and each part is flexible and universal. Therefore, there are many possible future applications, for instance, studying movement of NP-linked membrane molecules on membrane, endocytosis dynamics of NP-labeled receptors, dynamic analysis of intracellular NPs and vesicle-coated NPs transportation along m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CTAB coated gold nanorods(CTAB-AuNRs)	Nanoseedz	NR-40-650	85 nm * 40 nm
Color CMOS camera	Olympus	DP74	Japan
Coverslips	Citoglas	z10212222C	22*22 mm
Dark-field microscopy	Nikon	80i	upright microscope
Fetal bovine serum (FBS)	Gibco	10099141
Fiji	National Institutes of Health	2.0.0-rc-69/1.52 p	a distribution of ImageJ
Grooved glass slide	Sail brand	7103	Single concave
Image J	National Institutes of Health	1.52 j
MATLAB	MathWorks	R2019b
MATLAB Code			https://github.com/fenggeqd/JOVE-2020
Minimum essential medium (MEM)	Gibco	10-010-CVR	with phenol red
Minimum essential medium (MEM)	Gibco	51200038	no phenol red
Origin	OriginLab	Origin Pro 2018C
Penicillin-streptomycin	Gibco	15140122
Plastic cell culture dishes	Falcon	353002
Plastic cell culture dishes	Falcon	353001	35*10 mm
U87 MG cell	American Type Culture Collection	ATCC HTB-14	a human primary glioblastoma cell line