Our protocol uses single particle checking analysis to monitor the dynamics the location and the orientation and characterizes the diffusion of golden nanorods on cell membranes. Using this method, both the translational and the rotational dynamics of golden nanorods can be obtained and the dynamic can be comprehensively analyzed and the presented. This protocol has the potential to be used for the study of other types of complex biological systems.
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 two 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 five 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 darkfield microscope stage. For single particle tracking by darkfield microscopy, place a drop of oil onto the oil immerse 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 plugins, particle tracker classic, and particle tracker to open the particle detection and particle linking window and set the radius to six, the cutoff to zero, 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 matched, 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. To find the center pixel coordinate of the gold nanorod in each frame according to the XY coordinate, use the xycoordination. m function.
To delimit a three by three pixels matrix, extract the nine scattering intensity values of the red or green channels and calculate an average value, use the rgextraction. m function. Then use the polarangle.
m function to calculate the polar angles using the dual channel differential method. To calculate dynamic parameters using the formulas in the table, run the two analysis scripts. For visual analysis of the trajectory, set the X coordinate as X, the Y coordinate as Y, and the time as Y, then click plot, scatter, and color map, set the graph parameters, and add the color bar.
To generate mean square displacement time interval figures, set the time interval as X and the mean square displacement as Y.Click plot, scatter, analysis, fitting, nonlinear curve fitting and open dialogue and set the graph parameters. For multi-particle statistical analysis, set the dynamic parameters of interest as Y and click plot and histogram. Double-click the histogram to set the division size or the number of divisions and click apply.
Then add a column and set the graph parameters. For a time series analysis, set the time as X and the time series parameters as Y.Click plot, multi-pane, and stack. In the popup window, select line and OK.Then set the parameters for the graph.
The longitudinal plasmonic maximum of 40 by 85 nanometers CTAB coated gold nanorods is approximately 650 nanometers and the transverse resonance is 520 nanometers. The scattering intensity of the CTAB coated gold nanorods on U87 cell membranes demonstrates a typical Gaussian distribution with a narrow width consistent with that of CTAB coated gold nanorods on glass indicating that CTAB coated gold nanorods tracked in this experiment are well monodispersed. As illustrated, more than 500 CTAB coated gold nanorod trajectories can be tracked by darkfield microscopy and can be divided into long range diffusion trajectories and confined diffusion trajectories.
The radius of gyration of all 500 trajectories in this representative analysis showed a small value distribution with a mean radius of gyration of 0.5 micrometers and the max displacement was more distributed at small values. As demonstrated in this ensemble time average mean square displacement analysis, the CTAB coated gold nanorods normally diffuse with an alpha of approximately one. The density distribution of the diffusion coefficient and the alpha obtained from all of the trajectories, however, reveals that the dynamic of the gold nanorods exhibits a heterogeneous distribution with superdiffusion, Brownian, and subdiffusion motions.
In addition, single particle statistical analysis and time series parameter analysis can be used to characterize two representative long-term confined and moving trajectories. It is necessary to summarize and extract a single novel interpretation of data from on the single particle tracking analysis readout as there are typically some difficult tradeoffs to make.
