Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

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.

Begin by burning an ethanol-dipped glass coverslip on the flame, and placing the coverslip in a 35 by 10-millimeter cell culture dish containing 2 milliliters of cell culture medium without phenol red. Add 50 microliters of the cell suspension of interest onto the coverslip, and gently shake the dish back and forth and left and right to evenly distribute the cells.

Place the dish into the cell culture incubator for approximately 12 hours until the cells reach 20% to 40% confluency before adding 20 microliters of CTAB-coated gold nanorods to the dish. After gentle shaking to disperse the nanorods evenly across the dish, place the dish in a humidified atmosphere for 5 minutes.

At the end of the incubation, slowly transfer 100 microliters of supernatant from the dish into the groove of a glass slide, and carefully place the coverslip cell-side-down onto the slide groove. Then, seal the edge of the coverslip with nail polish and let the nail polish dry before placing the slide onto the dark-field microscope stage.

For single-particle tracking by dark-field microscopy, place a drop of oil onto the oil-immersed darkfield condenser and turn the knob until the condenser contacts the glass slide. Place a drop of oil onto the top of the cover glass and turn the focusing knob until the 60x oil immersion objective touches the oil.

Turn on the light source, and slightly turn the focusing knob to focus the imaging plane. Then, click the camera icon in the microscope software to record a sample scattering light image time-series using the color CMOS camera and save the image in a TIFF format.

To extract a single long-term trajectory, open the image in ImageJ and click Image | Type | and 8-bit to convert the image from RGB mode to 8-bit mode. To adjust the contrast, click Image | Adjust | Brightness | Contrast, and set the parameters. Select a target particle and use "Control + X" to cut off the time-series background.

Click Plug-ins | Particle Tracker Classic | and Particle Tracker to open the particle detection and particle linking window and set the radius to 6, the cutoff to 0, and the percentile to 0.01%. Set the link range to 10 and the displacement to 10, and click "OK" to open the particle tracker results window to see the results.

Click "Visualize All Trajectories" to inspect the generated trajectories. If the software-generated trajectory and the moving trajectory of the gold nanorods are a match, click "Save Full Report" to save the results. If the software-generated trajectory does not match the moving trajectory of the gold nanorods, click "Relink Particles" to relink the detected particles with different link range and percentile parameters.