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Resolving subdiffraction-limit distances

Plasmon coupling facilitates detection of direct contacts between optically colocalized gold nanoparticles in optical wide-field microscopy.
9 February 2009, SPIE Newsroom. DOI: 10.1117/2.1200902.1452

The famous quote by Yogi Berra, “You can observe a lot by watching,” undoubtedly applies to biological imaging if only slightly modified: “You can observe a lot by watching specifically labeled molecules with high temporal and spatial resolution.” Motivated by the idea that the location and motion patterns of individual molecules contain information about their fundamental biological functions, single-particle tracking has been used successfully to probe a variety of fundamental mechanisms, ranging from endocytosis1 to cell signaling.2 Conventional particle tracking cannot resolve subdiffraction-limit distances between identically labeled species. This is a significant constraint since interactions between identical components are common in important biological processes.

Although spectrally distinct probes can be colocalized with high spatial resolution,3 discrimination of identically labeled molecules is challenging because of the diffraction limit. Sophisticated ultraresolution fluorescent microscopy (fluorescent nanoscopy) is currently being explored4 to overcome this limitation. This technique is based on controlling the spatial excitation pattern, as done in stimulated-emission-depletion (STED) microscopy5 or by single-molecule localization of photoswitchable fluorophores. The latter has been realized by stochastic optical-reconstruction microscopy (STORM)6 and fluorescence-photoactivation-localization microscopy (PALM).7 Fluorescent nanoscopies are subject to the limited photostability and sensitivity of conventional fluorescent microscopy. Organic dyes blink and bleach, which limits the maximum observation time and complicates our analysis of single-dye trajectories. In addition, simultaneous tracking of individual components with high spatial and temporal resolution remains challenging.

Figure 1. Surface-confined gold nanoparticles (left) and spectral signatures (right) as a function of interparticle distance. (a) For interparticle separations Δ′ greater than the particle diameter, D, the near-field particle interaction is small and the resonance wavelength, λres, corresponds to that of an individual particle. (b) For Δ′′<D the plasmons in the individual particles couple and λres redshifts with decreasing separation. This is observable as an increase in the 580nm/530nm intensity ratio, R=I580nm/I530nm. (Reprinted with permission.8 ©2008 American Chemical Society.)

We propose to resolve subdiffraction-limit interactions through distance-dependent plasmon coupling between individual diffusing noble-metal nanoparticles.9–12 The plasmon-resonance wavelength (λres) redshifts with decreasing interparticle distance.13,14 DNA- and RNA-tethered pairs of gold nanoparticles can act as dynamic molecular rulers.15,16 Advantages of noble-metal probes include high optical cross sections and superb photostability. Nanoparticles do not blink and (in principle) allow continuous data acquisition without limitation of observation time.

Figure 2. Experimental setup. Individual gold nanoparticles are tracked in an inverted dark-field microscope. The light is chromatically separated, narrow-bandpass filtered (580 BP10 and 530 BP10, where BP10 indicates the bandpass in nanometers), and captured on an electron-multiplying charge-coupled device (EMCCD). (Reprinted with permission.8 ©2008 American Chemical Society.)

Figure 3. Ratiometric detection of interparticle-distance changes. (a) I580nm(red), I530nm (green), and total intensity (black) during collapse of a DNA-tethered gold-nanoparticle dimer (in arbitrary units, a.u.). (b) Corresponding intensity ratio R=I580nm/I530nm. Dimer compaction leads to a significant redshift of the plasmon resonance. (Reprinted with permission.8 ©2008 American Chemical Society.)

Figure 4. Point-spread functions (0.1s integration time) of two gold nanoparticles (P1 and P2) bound to the surface of an immortal cervical-cancer ‘HeLa’ cell before (a) and during (b, c) colocalization. The top and bottom rows show the image surface recorded at 530 and 580nm, respectively. The time of initial colocalization is set to t=0s. In (a) P1 and P2 are still discernable, while in (b) the two particles are superimposed. In (c) both the total intensity and R reach their maxima. (Reprinted with permission.8 ©2008 American Chemical Society.)

Plasmon-coupling microscopy combines conventional particle tracking with ratiometric analysis of the scattered light to detect spectral shifts caused by plasmon coupling of individual particles. Two-color tracking enables redshift detection in λres between nearby particles as changes in the particles' 580nm/530nm intensity ratio, R=I580nm/I530nm (see Figures 1 and 2) and thus provides information about direct near-field noble-metal nanoparticle interactions. In a first set of calibration experiments we assembled dimers of 40nm gold nanoparticles and monitored the ratio of the light scattered off the individual dimers while we induced dimer compaction. Collapse of a gold-nanoparticle dimer leads to a sudden increase in total scattering intensity and a strong redshift (as indicated by the increase in R: see Figure 3).

We subsequently monitored interactions between gold-nanoparticle-labeled surface receptors on HeLa cells (an immortal cervical-cancer cell line) using plasmon-coupling microscopy. This approach is sensitive to distances of tens of nanometers, i.e., significantly below the diffraction limit. Figure 4 shows the curve-fitted images (point-spread functions) for two particles (P1 and P2) at three time points during aggregation. The higher total intensity of P1 compared to P2 indicates that P1 is larger. Initially the intensities of both P1 and P2 are higher in the green than the red channel. This changes when the particles approach each other sufficiently closely for the particle plasmons to couple so that the resonance wavelength redshifts. In Figure 4(b) the particles are no longer optically discernible. Concurrently with the optical colocalization the intensity distribution across the detection channels reverses. The peak intensity is now significantly higher at 580 than at 530nm, and R has increased to R=1.3. In Figure 4(c) both the total intensity and R reach their maxima. The high R=1.6 reveals strong interparticle coupling.

We combined single-particle tracking with ratiometric wavelength detection to observe near-field interactions between individual gold nanoparticles.8 This method can resolve distances among identical surface groups on nanometer scales as a function of time and could become a useful tool to study a broad range of cell-surface processes where colocalized surface species undergo dynamic or transient interactions that are otherwise hidden by the diffraction limit. Future challenges include a further reduction in probe size, improvement of the particles' stability in biological buffers, and obtaining control over the number of functional groups on the labels to prevent cross-linking of surface receptors.

This work was supported by grant 1 R21 EB008822-01 from the National Institutes of Health.

Björn M. Reinhard
Department of Chemistry
The Photonics Center
Boston University
Boston, MA

Björn M. Reinhard was educated in Germany. He received his diploma in chemistry from the Technical University of Munich and his doctorate from the Technical University of Kaiserslautern in 2004. From 2004 to 2006 he was a postdoctoral researcher at the University of California at Berkeley, where he worked with Jan Liphardt and Paul Alivisatos. He joined Boston University in 2007.

Hongyun Wang, Guoxin Rong, Lynell Skewis
Department of Chemistry
Boston University
Boston, MA

Hongyun Wang earned her BS from the University of Science and Technology of China, where she worked with Shilin Liu. After her graduation in 2007 she joined the Reinhard laboratory, where she is currently developing optical tools to study fundamental biological processes at the single-molecule level.

Guoxin Rong obtained his BS from the University of Science and Technology of China working with Yi Xie. In 2007 he became a PhD candidate in the Reinhard laboratory. His research interests focus on the development and application of plasmon-coupling microscopy.

Lynell Skewis received her BS in chemistry from the University of Washington in 2006. She is a PhD candidate supported by a Ruth L. Kirschstein National Research Service Award predoctoral fellowship from the National Institutes of Health.