eoe sub articles

EoE Sub Articles

1. Microscope slide preparation
NOTE: U87 MG cells of the third to tenth generation with high activity are used in SPT experiments.
<ol>
	<li>Sterilize 22 mm &#215; 22 mm coverslips already cleaned with Piranha solution by immersing in ethanol (99.9%).</li>
	<li>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 (...

darkfield microscopy to visualize diffusional dynamics of nanorods on cell membrane

Source: Ge, F., et al. <a href="https://app.jove.com/v/61603">Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21458/21458_Figure1.jpg" /> 
Figure 1: Schematic diagram of the optical path of darkfield microscopy, microscope slide preparation, and the dual-channel difference method for calculating polar angle (&#952;) of AuNRs. Scale bar in image is 2 &#956;m.
<img alt="Figure 2" src="/files/ftp_upload/21458/21458_Figure2.jpg" /> 
Figure 2: Characterization of CTAB-AuNRs. (A) TEM images of CTAB@AuNRs....

Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

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

To investigate gold nanorod diffusional dynamics on a live cell membrane using darkfield microscopy, DFM, combined with single-particle tracking, begin with a sterilized coverslip in a culture dish containing suitable medium. Add the cell suspension onto the coverslip. The medium facilitates cell growth.
Incubate with positively-charged gold nanorods that adsorb onto the negatively-charged cell membrane via electrostatic interactions. Pipette the medium from the dish to the groove of a glass slide. Place the coverslip cell-side-down on the groove, and seal.
Place the slide on the darkfield microscope stage. The microscope illuminates the sample with a high-angle oblique light passing through a specialized darkfield condenser.
Upon the illumination of the nanorods, the free electrons on the nanorod surface oscillate at a frequency resonant with the incident light. The oscillating electrons scatter light at a specific wavelength captured by the objective lens, enabling nanorod visualization.
Similarly, the cell membrane &#8212; composed of lipids and proteins &#8212; scatters the incident light at a higher refractive index than the surrounding medium, creating a bright image against a dark background. Capture time series DFM images.
Using a suitable software, perform single-particle tracking analysis on the DFM images to visualize the diffusion of the nanorods through the cell membrane with high spatiotemporal resolution.

Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

In This Article

Overview