Colocalization of individual components is often used in biological imaging as an indication for direct interactions between the labeled components. However, due to the diffraction resolution limit, individual components can be hundreds of nanometers apart and still appear colocalized in conventional light microscopy. In our set-up we used a 60x objective with a NA = 0.65. Under these conditions two individual gold nanoparticles with λres
≈ 535 nm can be discerned optically only if they are separated by more than 500 nm. Plasmon coupling microscopy now offers additional information about very short interparticle separations by detecting the near-field interactions between individual noble metal nanoparticle labels which occur only if the particles have approached each other to within approximately one particle diameter. We applied this approach to detect direct interactions between individual nanoparticle labeled integrin surface receptors on living cells during colocalization. Integrins are a family of cell surface receptors that mediate a series of cell-cell and cell-matrix interactions, for instance with the cell adhesion molecule fibronectin.47–50
We bound fibronectin to integrins on the plasma membrane of a cervical cancer cell line (HeLa) and used 40 nm gold nanoparticles that were functionalized with anti-fibronectin (see Supporting Information
) as labels for the integrin bound fibronectin. All experiments were performed in a flowchamber at 37 °C.
In a typical experiment the cells were first incubated with a 0.2 mg/mL solution of fibronectin in imaging buffer for 10 minutes. Then the cells were washed with an excess of imaging buffer and a solution of anti-fibronectin functionalized gold nanoparticles in imaging buffer was added. The concentration of the particles was chosen sufficiently low to allow the tracking of individual particles. We started recording with the addition of gold nanoparticles and detected gold nanoparticle binding from solution in real time. Since the cells always contained some particulate scattering sources we restricted our analysis to gold nanoparticles whose binding from solution was recorded. To confirm that these attachments were caused by specific interactions between the fibronectin and the gold nanoparticle bound antibody, we performed different control experiments. First, we used bovine serum albumin (BSA) functionalized nanoparticles to test the “stickiness” of the cell surface for protein coated nanoparticles. We observed no binding under our experimental conditions. Next, we incubated anti-fibronectin functionalized particles with HeLa cells without prior incubation with fibronectin. Again we did not observe any particle binding, giving confidence that the observed binding was not caused by non-specific membrane – particle interactions but was indeed caused by the antibody binding to its epitope.
To track particles that bound to the cell from solution and to monitor their R
value, we fitted the scattering images on the two color channels.41
The obtained fits were then background corrected with fits to nearby areas that were void of nanoparticles. We used the fitted peak intensities on the two color channels I580nm
to calculate the ratio R = I580nm/I530nm
for the individual particles. The spatial coordinates of the individual particles were obtained from the fitted peak position on the 530 nm channel. The surface-mobilities of the nanoparticle labels varied significantly, the observed behaviors varied from immobilization to rapid surface diffusion with diffusion coefficients up to 1.8×10−14
/s. It has been observed before that the lateral mobility of individual integrins can vary substantially.51,52
The mobility of the gold nanoparticle labeled fibronectin-integrin complexes on the cell surface depends on the oligomerization state of the integrin and the interactions with the cytoskeleton of the cell.47,48,51,53
We observed that some of the tracked gold nanoparticle labeled fibronectin-integrin complexes approached each other to within the diffraction resolution limit of our microscope so that we could no longer resolve the individual particles optically. Colocalization durations ranged from a single frame to hundreds of frames, in some cases the particles remained colocalized after initial contact for the entire remaining observation period. While for very short colocalization durations we assumed that the vicinity was accidental, different processes can cause longer periods of colocalization. It is conceivable that gold nanoparticle labeled fibronectin-integrin complexes cluster due to direct or mediated short-range interactions. However, alternative processes exist that can account for the optical colocalization which don’t require direct interactions between the integrins. For instance, it has been observed before that membrane spanning proteins can be locally stopped, slowed or temporarily confined due to the compartmentalization of the cell membrane.54
These compartments can have dimensions on the order of the diffraction resolution limit or below. If two nanoparticle labeled fibronectin-integrin complexes get temporarily trapped in the same compartment, they appear colocalized for the time they remain in the same compartment.
Plasmon coupling offers valuable additional information about the interparticle separation during colocalization; it enables to experimentally probe interactions between the particles that occur only on the tens of nanometer length scale and is therefore a promising tool to unravel the interactions between gold nanoparticle labeled surface receptors. contains an example that illustrates how short range interactions between individual gold nanoparticles can be detected by plasmon coupling on a cell surface. We show the curve fitted images, or point-spread-functions (PSF), for the two particles at three time points during aggregation. In the two particles are still discernable. It is striking that one of the particles (P1) is brighter than the other particle (P2). For both P1 and P2 the intensity is higher on the green than on the red channel, albeit the computed R values imply that λres for P1 is redder than for P2. The differences in the optical properties of P1 and P2 indicate differences in the size and/or shape of the particles. P1 could be a larger spherical particle, an anisotropic particle such as a gold rod, or a small cluster of gold nanoparticles (for instance a dimer). Independent of the exact nature of the individual particles, we observe that their spectral features change when they approach each other. In P1 and P2 are no longer optically discernable; concurrent with the colocalization both the total intensity and the intensity distribution on the two channels change. The peak intensity is now significantly higher on the 580 nm than on the 530 nm channel, and the intensity ratio has increased to R = 1.3. In both the total intensity and the R value reach their maximum. The high R value of R = 1.6 reveals strong interparticle coupling. We continued monitoring of the particles for another 13 minutes and did not observe P1 and P2 to separate again. The observed strong spectral shifts together with the prolonged colocalization are indications for non-reversible short-range interactions between the nanoparticle labeled fibronection-integrin complexes in this case.
Figure 5 Point spread functions with 0.1 s integration time of two gold nanoparticles (P1 and P2) bound to the surface of a HeLa cell before a) and during colocalization (b) and (c). The top row shows the fitted surface of the image recorded on the 530 nm channel, (more ...)
We point out that in the case of the investigated gold nanoparticle labeled fibronectin-integrin complexes prolonged colocalization was not necessarily correlated with a sustained spectral shift. Instead, in many cases we observed fluctuations in the R
values on short and long time scales, indicating some dynamic in the average interparticle separations during colocalization. This is illustrated for a pair of particles in . Approximately 10 s after the second particle had bound to the cell surface, the two particles “collided” and remained colocalized for over 100 s. In we show the interparticle separation as obtained from the fitted PSFs and the computed R
values before and after the collision; the continuous red line in indicates a 5 point sliding average of R
. Shortly after colocalization we observe an increase in the average R
value from 0.55 to 0.75 in two steps. A first step occurs at t = 1.9 s and a second more prominent follows at t = 16.5 s. The R
level remains at R
= 0.75 during the interval t = 16.5 s – 25.5 s. Then it drops in two steps, first to R ≈ 0.6 at t = 25.5 s and then to R ≈ 0.55 at t = 82.2 s. At the end of the trajectory the R
baseline has reached again a level close to that of the separated particles. The observed fluctuations imply some flexibility in the average interparticle separation. This finding together with the moderate shift in R
upon colocalization, implies that the two particles are not non-reversible bound to each other but remain separated. One possible explanation for the behavior observed in is a confinement of the two gold nanoparticle labeled fibronectin-integrin complexes in one membrane compartment.54,55
In case the confined space is sufficiently small, the time average of λres
red-shifts due to plasmon coupling between the interacting nanoparticles. Variations in the dimensions of the compartment change the average interparticle separation and therefore lead to systematic shifts in the time average of the plasmon resonance wavelength. Fluctuations of λres
on faster time scales result from point-to-point variations of the interparticle separation within the compartment.
Figure 6 a) Separation of the point spread function centroids and b) R = I580nm/I530nm values for two gold nanoparticle labels diffusing on the surface of a HeLa cell as function of time. The continuous red line in b) is a 5 point sliding average. The two particles (more ...)
In most cases colocalization lasted much shorter than shown for the examples in and . shows an example of a temporary colocalization of two individual gold labels with representative duration. The particles remain colocalized for approximately 7s, then they separate and become optically discernable again. The calculated R values during colocalization show no noticeable differences to the R values of the individual gold nanoparticles prior and after colocalization. For one of the particles (Particle 2) R even slightly decreases upon colocalization. We ascribe this effect to an increase in signal-to-noise; colocalization of two noninteracting gold nanoparticles leads to a gain in “green light” scattered from the diffraction limited spot. Experiments like the one shown in and others in which we tracked individual particles that showed a constant average R value as function time revealed that the refractive index at the cell/medium interface is constant under our experimental conditions.
Figure 7 a) Separation of the point spread function centroids and b) computed intensity ratios R = I580nm/I530nm for two gold nanoparticle labels, Particle1 and Particle2, that temporarily colocalize as function of time. The point of initial optical colocalization (more ...)
In summary, our investigations of the interparticle separation of colocalized gold nanoparticle labels using plasmon coupling microscopy show that the nature of the particle interactions during optical colocalization can vary significantly. We observed strong coupling between the gold labels as well as non-interaction even in the case of prolonged colocalization. These findings underline the insufficiency of the colocalization criterion for the unambiguous detection of short-range interactions in particle tracking and the value of plasmon coupling microscopy to overcome this shortcoming.