Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

Abstract

Source: Ge, F., et al. Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy. J. Vis. Exp. (2021).

In this video, we describe the darkfield microscopy (DFM) technique combined with single-particle tracking (SPT) analysis to monitor the interaction of plasmonic gold nanorods with the cell membrane of a specific cell line of interest. Combined DFM and SPT analysis allows the visualization of the nanorod movement through the cell membrane with high spatiotemporal resolution.

Protocol

1. Microscope slide preparation

NOTE: U87 MG cells of the third to tenth generation with high activity are used in SPT experiments.

1. Sterilize 22 mm × 22 mm coverslips already cleaned with Piranha solution by immersing in ethanol (99.9%).
2. Use forceps to take out the cover slip from the ethanol solution (step 1.1) and sterilize by burning ethanol on the flame. Once all ethanol is burnt, place coverslips in a plastic cell culture dish (35 x 10 mm) filled with 2 mL of cell medium (no phenol red).
3. Add 50 µL of the cell suspension on the coverslip and gently push the dish....

Results

Figure 1: Schematic diagram of the optical path of darkfield microscopy, microscope slide preparation, and the dual-channel difference method for calculating polar angle (θ) of AuNRs. Scale bar in image is 2 μm.

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	Image J	1.52 j
MATLAB	MathWorks	R2019b
MATLAB Code			https://github.com/fenggeqd/ JOVE-2020
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
Minimum essential medium (MEM) 	Gibco 	51200038	No phenol red
Penicillin-streptomycin 	Gibco 	15140122