Encyclopedia of Experiments: Biological Techniques
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Transcript
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 — composed of lipids and proteins — 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.
