Tracking Biomolecules and Nanoparticle Probes in Biological Samples with Improved Microscopy Imaging Methods

Jan 12, 2016
By Spectroscopy Editors

Novel optical imaging methods have opened new frontiers in chemical and biological discovery because they are minimally intrusive, requiring virtually no contact with the sample. Ning Fang, who is an associate professor of chemistry at Georgia State University, specializes in single-molecule and nanoparticle imaging, bioanalytical chemistry, and biophysics. He recently spoke to us about his work with far-field optical imaging methods for visualizing the dynamics of biomolecules and nanoparticle probes.







Your laboratory’s efforts largely have been based on using differential interference contrast (DIC) microscopy as an imaging technique for chemical and biological research. Briefly, how does it work?

DIC microscopy has long been used as a complementary technique to image cells because it provides better visualization (higher contrast, better resolution, and shallower depth of field) of cellular features than other far-field optical microscopy techniques. With the funding support from the U.S. Department of Energy (DOE), the recent efforts in the Fang laboratory have transformed DIC microscopy into a primary research tool for tracking plasmonic nanoparticles in biological samples.

The Nomarski DIC microscope is the primary microscope design for imaging in the visible and near-IR range. In this design, the microscope is equipped with two polarizers and two Nomarski prisms. The first Nomarski prism splits the illumination beam into two orthogonally polarized beams that are separated by a sub-wavelength shear distance. Two intermediate images are generated behind the microscope objective and then are laterally shifted back by the second Nomarski prism to overlay and form the interference DIC image. In essence, the Nomarski DIC microscope works as a two-beam interferometer that detects the optical path difference of the two beams passing through the specimen. Under a DIC microscope, anisotropic gold nanorods display disproportionate bright and dark spots depending on their orientation relative to the optical axes and the illumination wavelength.

One of your group’s publications discusses the use of DIC microscopy to examine the dynamic rotational motion and orientation of gold nanorod probes in live cells (1). What other microscopy techniques have been used for this purpose, and how is the DIC technique an advancement?

Other optical microscopy techniques include the scattering-based dark field microscopy, planar illumination microscopy, total internal reflection scattering microscopy, and the absorption-based photothermal heterodyne imaging.

Imaging under a DIC microscope benefits from its minimal invasiveness to biological samples with low illumination light power and no fluorescence staining, less interference from the background scattering, and its ability to take images of both nanoparticle probes and cellular features simultaneously.

What are the dimensions of the gold nanorods, and how are they modified so they can bind to the cells?

The gold nanorods used in our imaging experiments have a length of 30–120 nm and an aspect ratio (length:width) of 2–4. The longitudinal plasmon resonance wavelengths of these particles are within the visible and near-IR ranges.

The functionalization of gold nanoparticles has become routine with well-established methods reported in the literature (2,3). Gold nanorods have been functionalized with various surface modifiers before they were used in imaging experiments in my laboratory (4). For example, a thiol-exchange method was used for the PEG-modified nanorods. A bifunctional linker, N-hydroxylsuccinimide-PEG-SH, was used for transferrin-modified nanorods and cell-penetrating peptide TAT-modified nanorods. A polyethylenimine (PEI)-modified nanorod was created using a thiol-exchange step first performed with 11-mercaptoundecanoic acid, followed by adsorption of the cationic PEI polymer onto the surface via electrostatic attractions.

Your laboratory more recently developed the single-particle orientation and rotational tracking (SPORT) technique, which is based on the DIC microscopy approach. Briefly, how does this technique differ from DIC microscopy and what types of information does it allow you to obtain?

DIC microscopy has been used mostly as a secondary imaging technique to provide complementary information to the fluorescence-based techniques since its invention in 1950s. Part of the reason for this situation is the subtle operation that is dictated by the principle of interferometry to obtain reproducible images and quantitative measurements. Our efforts have been focused on establishing DIC microscopy as a major imaging technique for chemical and biological research.

The SPORT technique is essentially the combination of DIC microscopy, plasmonic gold nanorods, and a host of data analysis methods to extract single particle dynamics. Some features of SPORT include

• High angular resolution (1° in optimized cases)

• Resolving 3D orientation and enabling dynamic 5D tracking

• Identification of fundamental rotational modes (in-plane and out-of-plane rotations)

• Analysis of rotational rate and mode with a millisecond temporal resolution

Another publication from your group describes the use of the SPORT technique to study the transport of gold nanorod cargos in the axons of live nerve cells (5). What did this study tell you about the transport process within the cells? What other biological processes could this approach be used to study?

Reference 5 discusses how the high-speed SPORT technique was developed for direct visualization of the rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport with a temporal resolution of 2 ms. The cargo’s rotational dynamics at pauses is the direct consequence of forces applied, disclosing the status of motor protein competition and possible regulation by associate proteins. We discovered that the cargos have sufficient freedom to perform random in-plane rotations but remain tethered to the microtubule track by motor proteins or other associated proteins such as dynactin. In addition, when molecular motors encounter obstacles, such as other cargos, microtubule-associated proteins, and intersecting microtubules, they may pause until the track becomes available again to continue the transport, or they may overcome these obstacles by switching to other microtubule tracks.

Another important biological process that we are currently studying is endocytosis. We are trying to visualize the rotational motions induced by endocytic macromolecular nanomachines in live cell membranes. Hundreds of proteins are involved in the complex endocytic pathways, and their physical motions and biological functions are not understood comprehensively. For example, clathrin-mediated endocytosis has been studied extensively by electron and fluorescence microscopy, but it is still unclear how cargos rotate during different stages of internalization and how the rotational motions coincide with molecular activities, such as dynamin assembly cutting at the neck of clathrin-coated pits.

What are you currently studying, and what are the next steps in your research?

The most recent development of SPORT (the FACSS award, see reference 6) allows us to reliably track gold nanorods in 5D (x, y, z, and two orientation angles). The next step in instrument development is to combine SPORT with other optical imaging modes, including light sheet microscopy and super-resolution fluorescence microscopy. The ability to correlate the motions of nanoparticles with the location and organization of fluorescent biomolecules that directly participate in the specific motion will reveal detailed molecular mechanisms involved in both the internalization and intracellular transport of nanoparticle cargos. The emphasis is the ability to either acquire images in different modes simultaneously or switch between different modes very rapidly to accomplish fast imaging speed.

On the other hand, we are more focused than ever to push for important biological discoveries. My lab is funded by National Institutes of Health (NIH) to acquire new fundamental knowledge about the detailed rotational dynamics of cellular membrane processes, such as adhesion, transport, and endocytosis of functionalized nanoparticles, as may be relevant to drug delivery and viral entry. The rotational patterns on cell membranes for functionalized gold nanorods will be identified and correlated with their lateral movements and the presence of relevant functional biomolecules tagged with fluorescent proteins. The characteristic rotational motions of cargos during different internalization pathways will be visualized directly, leading to new opportunities for understanding the timing, signaling, and chemical and mechanical functions of protein modules involved in different pathways. Computer simulations will also be developed to understand the effects of nanoparticle shapes, sizes, and surface modifiers. We are hoping our research may initiate a shift in the current research paradigm on membrane structure and function by demonstrating the importance of rotational dynamics at the single molecule and nanoparticle level.


(1) G. Wang, W. Sun, Y. Luo, and N. Fang, J. Am. Chem. Soc. 132(46), 16417–16422 (2010).

(2) X. Huang, S. Neretina, and M.A. El-Sayed, Adv. Mater. 21, 4880 (2009).

(3) E. Locatelli, I. Monaco and M. Comes Franchini, RSC Adv. 5, 21681 (2015).

(4) Yan Gu, Wei Sun, Gufeng Wang, and Ning Fang, J. Am. Chem. Soc. 133, 5720 (2011).

(5) Y. Gu, W. Sun, G. Wang, K. Jeftinija, S. Jeftinija, and N. Fang, Nature Communications (2012), DOI: 10.1038/ncomms2037.


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