This work was supported by the National Natural Science Foundation of China with grant numbers of 21425519, 91853105 and 21621003.

1. Cell culture
<ol>
	<li>Prepare complete medium for U87 MG cells by adding fetal bovine serum (final concentration 10%) and penicillin-streptomycin (final concentration 1%) to the minimum essential medium (MEM). Use plastic cell culture dish for cells subculture.</li>
	<li>Passage cells 2 to 3 times a week.
	<ol>
		<li>Remove the culture medium and rinse the cell layer with Dulbecco&#39;s phosphate-buffered saline (D-PBS) 2~3 times when confluent (80%~90%).</li>
		<li>Add 1.0 to 2.0 mL of Trypsin-EDTA solution to the...

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The authors have nothing to disclose.

The presented protocol is used to study the dynamics of AuNRs on cell membrane. The protocol consists of four parts, including microscopic imaging, data extraction, dynamic parameters calculation and data analysis methods, and each part is flexible and universal. Therefore, there are many possible future applications, for instance, studying movement of NP-linked membrane molecules on membrane, endocytosis dynamics of NP-labeled receptors, dynamic analysis of intracellular NPs and vesicle-coated NPs transportation along m...

The dynamics of nanoparticles (NPs) on the membrane is closely associated with the cellular uptake process, which is essential for the understanding of cell functions, viral or bacterial infections and the development of artificial nanomedical delivery systems1,2. Single particle tracking (SPT) technique is a robust tool for characterizing the heterogeneous behaviors of NPs3,4. In general, cell membrane is fluidic, which means that the components such as proteins and lipids can move laterally in the plasma membrane plane5,6,7. The spatiotemporal complexity of membrane organization and structure may lead to spatiotemporal heterogeneity of the interaction between NPs and membrane. Hence, direct visualization of the movement of NPs on the membrane requires both high spatial and temporal resolution.
Single particle tracking microscopy that monitors the localization of individual particles in living cells with a spatial resolution of tens of nanometers and a time resolution of milliseconds has been well developed to study the NPs or membrane molecules dynamics8,9. Fluorescence-based microscopic imaging techniques have become valuable tools for observing NPs/molecules in living cell environment9,10,11,12. For example, total internal reflection fluorescence microscopy, which images thin layers (~100 nm) of the sample at the substrate/solution interface with a high spatiotemporal resolution has been widely used in studies of membrane molecules dynamics13,14. However, the inherent disadvantages of single fluorophores, such as low intensity and rapid irreversible photobleaching reduce the accuracy and duration of tracking13. Therefore, non-fluorescent plasmonic NPs, which replace the fluorescent probes, have attracted more and more attention in long-term imaging studies due to their unique optical characteristics15. Based on the scattering signals of plasmonic NP probes, several kinds of optical microscopic imaging technologies have been used to study the mechanism of biological processes, such as dark-field microscopy (DFM)16, interferometric scattering (iSCAT) microscopy17 and differential interference contrast microscopy (DICM)18. In addition, the motion and rotation dynamic of AuNRs can be obtained using DFM and DICM18,19,20,21,22. Typically, in an SPT experiment, the motion of the object is recorded by the optical microscope, and then analyzed by SPT analysis methods3. The time-resolved trajectories and orientational angles generated by individual NPs are normally stochastic and heterogeneous, so it is necessary to present abundant dynamic information with various analysis methods.
Here, we provide an integrated protocol that monitors the dynamics of AuNRs on cell membrane using DFM, extracts the location and orientation of AuNRs with ImageJ and MATLAB and characterizes the diffusion of AuNRs with SPT analysis methods. As a demonstration, we show here how to use the SPT protocol to visualize dynamics of unmodified AuNRs (CTAB-AuNRs, synthesized by cetyltrimethylammonium ammonium bromide molecule as protective agent) on U87 MG cell membrane. It has been demonstrated that CTAB-AuNRs can adsorb proteins in biological environment, move on cell membrane and then enter cells2,20,22. U87 MG cell is the most common and most malignant tumor of the central nervous system, and its membrane receptors are abnormally expressed. The membrane receptors can interact with proteins on AuNRs, which influence the dynamics of AuNRs. Our protocol is generally applicable to other SPT experiments in the field of biology.

<table>
	<tbody>
		<tr>
			<td>CTAB coated gold nanorods(CTAB-AuNRs)</td>
			<td>Nanoseedz</td>
			<td>NR-40-650</td>
			<td>85 nm * 40 nm</td>
		</tr>
		<tr>
			<td>Color CMOS camera</td>
			<td>Olympus</td>
			<td>DP74</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Citoglas</td>
			<td>z10212222C</td>
			<td>22*22 mm</td>
		</tr>
		<tr>
			<td>Dark-field microscopy</td>
			<td>Nikon</td>
			<td>80i</td>
			<td>upright microscope</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health</td>
			<td>2.0.0-rc-69/1.52 p</td>
			<td>a distribution of ImageJ</td>
		</tr>
		<tr>
			<td>Grooved glass slide</td>
			<td>Sail brand</td>
			<td>7103</td>
			<td>Single concave</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institutes of Health</td>
			<td>1.52 j</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2019b</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Code</td>
			<td></td>
			<td></td>
			<td>https://github.com/fenggeqd/JOVE-2020</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>10-010-CVR</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco</td>
			<td>51200038</td>
			<td>no phenol red</td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>OriginLab</td>
			<td>Origin Pro 2018C</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cell culture dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>35*10 mm</td>
		</tr>
		<tr>
			<td>U87 MG cell</td>
			<td>American Type Culture Collection</td>
			<td>ATCC HTB-14</td>
			<td>a human primary glioblastoma cell line</td>
		</tr>
	</tbody>
</table>

visualizing diffusional dynamics of gold nanorods on cell membrane using single nanoparticle darkfield microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

In the protocol, the unmodified 40 x 85 nm CTAB-AuNRs were used. As shown in Figure 2B, its longitudinal plasmonic maximum at is ~650 nm (red region) and transverse resonance is at 520 nm (green region). Previous literatures have revealed that the optical properties (such as LSPR intensity) of plasmonic AuNRs will change significantly with their diameter20,22. In Figure 2C, the scattering intensity from ...

Watch this Scientific Journal Video about Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy at JoVE.com

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

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.

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JoVE Journal

Biochemistry

4.2K visualizações.  Tsinghua University. Nosso protocolo utiliza uma análise de verificação de partículas únicas para monitorar a dinâmica do local e da orientação e caracteriza a difusão de nanorods dourados nas membranas celulares. Utilizando este método, tanto a dinâmica translacional quanto a rotacional dos nanorods dourados podem ser obtidas e a dinâmica pode ser analisada de forma abrangente e apresentada. Este protocolo tem potencial para ser utilizado para o estudo de outros tipos de sistemas biológicos complexo...