Spatiotemporal activity patterns, displaying high spatial and temporal resolution, would allow for an improved quantitative description of tissue morphogenesis. There is a clear need for probes that are capable of high spatial and temporal resolution. Plasmon rulers, consisting of coupled gold nanocrystals, have the potential to play an important role as improved probes for measuring quantitative spatiotemporal activity patterns ().
Improved temporal resolution arises inherently from the physical properties of a gold nanocrystal. A gold nanocrystal exhibits a surface plasmon in the presence of an electromagnetic field. A surface plasmon is the collective oscillation of electrons at the interface between two materials, in this case, the metal nanocrystal and the dielectric local environment. The surface plasmon is most prominent at the resonance condition where the wavelength of the electromagnetic field is matched to the resonance wavelength of the nanocrystal, allowing for coupling to occur between the electromagnetic field and the electrons.45
The nanocrystal resonance wavelength depends on the shape, size, and local environment of the nanocrystal.46
This resonance can result in intense light scattering from the nanocrystal that is constant with respect to time. The light scattering spectrum is time-invariant and can be continuously acquired in real-time in a few milliseconds, resulting in high (ms) temporal resolution over the course of long imaging periods. Potentially as long as hours or even days, the total imaging time is not limited by the properties of the probe nanoparticle.
Improved spatial resolution arises from the physical mechanism of coupled gold nanocrystals. Two gold nanocrystals can be coupled together using a single biomolecule substrate (i.e., cleavable peptide substrate, peptide scaffold), where the substrate length determines the distance between the two nanocrystals. The substrate length is designed so that the distance between the two nanocrystals is less than their diameter. In this configuration, the surface plasmons of each nanocrystal couple together, resulting in a shift in the resonance wavelength. The strength of this surface plasmon coupling and the resulting wavelength shift is dependent on the distance between the two nanocrystals,23,47,48
and can be colorimetrically and spectroscopically observed (). In the event that cleaving or binding occurs to the biomolecule substrate, the distance between the two nanocrystals will change, which is observable in the light scattering spectrum. Since changes in surface plasmon resonance wavelength can be correlated with changes in distance,47,48
coupled gold nanocrystals, otherwise referred to as a plasmon ruler, can be used to observe a single cleaving or binding event.23,24,26,49-52
High spatial resolution is achievable because of the large scattering cross-section and therefore high intensity of the scattered spectrum, permitting the analysis of events at the single-molecule level.
Figure 4 Plasmon rulers. (A) Single gold nanocrystals and coupled gold nanocrystals imaged by illuminating with unpolarized white light and collecting scattered light using a darkfield microscope in transmission mode. (B) Plasmon ruler consists of two gold nanocrystals (more ...)
Using surface plasmon coupling, nanometer-scale interactions below the diffraction limit have been observed. The colocalization of individual integrin surface receptors on a cell membrane was visualized with sub-diffraction resolution using plasmon coupling between pairs of gold nanocrystals in two-dimensional human cervical carcinoma cell culture models.25
The cell adhesion molecule fibronectin was first bound to integrin surface receptors. Single gold nanocrystals, functionalized with anti-fibronectin, were then bound to the receptors by formation of fibronectin-integrin complexes (). Lateral movement of individual receptors was then tracked over time by intensity analysis of the ratio of the light scattering intensities at 530 nm and 580 nm. Individual receptors were initially separated. As the moving receptors co-localized, an increase in the ratio of light scattering intensities was seen due to coupling of surface plasmons of the bound nanocrystals (). Trafficking, clustering, and dimerization of other receptors, such as epidermal growth factor receptors, have also been imaged with sub-diffraction resolution using plasmon coupling.27,28,53,54
Figure 5 Receptor colocalization visualized at the single-molecule level. (A) Single gold nanocrystals bound to individual surface receptors. Individual receptors visualized at the single-molecule level. Colocalization of individual receptors visualized with subdiffraction (more ...)
Improved temporal resolution of plasmon rulers enables the ability to visualize activity in real time and resolve intermediate dynamics that otherwise would be very difficult to observe using conventional bulk methods with lower temporal resolution. Using plasmon rulers, an intermediate step in enzyme catalysis was captured recently at the single-molecule level.24
Plasmon rulers consisted of a gold nanocrystal dimer linked with DNA containing a single restriction site for the restriction enzyme EcoRV (). In order to capture enzymatic dynamics on the order of micro- to milli-seconds, changes in plasmon coupling were imaged by intensity analysis rather than spectral analysis. The scattering cross-section of a nanocrystal dimer depends on its interparticle distance. When the restriction enzyme cut the DNA, the nanocrystals separated, resulting in a decrease in the light scattering intensity. Using continuous imaging of single-particle trajectories, an intermediate step was captured where the enzyme, upon binding, momentarily bent the DNA. The bending of the DNA caused the two nanocrystals to become closer, resulting in a decrease in the interparticle distance and therefore an increase in the light scattering intensity (). The enzyme then cut the DNA and a decrease in the light scattering intensity was seen. The observation of these intermediate dynamics is made possible because of the high temporal resolution of plasmon rulers. In addition to nuclease activity, protease activity has similarly been imaged () using peptide-linked nanocrystals.26
As compared with fluorophore-based imaging methods, plasmon rulers do not suffer from photobleaching, and are capable of long-term and continuous imaging as demonstrated by long-term imaging of caspase protease activity in two-dimensional cell culture models.26
We expect that the improved temporal resolution of plasmon rulers should allow for measurement of intermediate dynamics in activity patterns ().
Figure 6 High temporal resolution of plasmon rulers resolves intermediate dynamics. (A) Plasmon ruler consists of a gold nanocrystal dimer linked with DNA containing a single restriction site for a restriction enzyme EcoRV. Intermediate events over time: The enzyme (more ...)
In conclusion, plasmon rulers bring significant improvements in spatial and temporal resolution and should enable measurement of more quantitative activity patterns. Complex three-dimensional assemblies are being developed to provide conformational information as well as distance dependence.55
Plasmon rulers are currently being developed for quantitative measurement of protease activity during tissue morphogenesis in three-dimensional culture models and in vivo. Global modulation with protease activators/inhibitors or local modulation by optical gene silencing55-58
of proteases can be also utilized to systematically modulate spatiotemporal activity patterns. We expect these models to lead not only to an improved quantitative description of tissue morphogenesis but possibly also to advance therapeutic strategies for the repair of diseased or damaged tissues.