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.
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	<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.