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.
1. Microscope slide preparation
NOTE: U87 MG cells of the third to tenth generation with high activity are used in SPT experiments.
2. Performing single particle tracking experiments with darkfield microscopy (Figure 1).
3. Data acquisition
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.
Figure 2: Characterization of CTAB-AuNRs. (A) TEM images of CTAB@AuNRs. (B) LSPR spectrums of CTAB@AuNRs. (C) Distribution of single particle intensity: CTAB@AuNRs on glass (left panel), CTAB@AuNRs on plasmon membrane (right panel).
Figure 3: Pretreatment of darkfield images. Time-series color images were converted to “8 bit” mode and their contrast were adjusted.
Figure 4: Extraction of single long-term trajectory with ImageJ.
The authors have nothing to disclose.
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 |