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Register a new userA highly specific gold nanoprobe for live-cell single-molecule imaging
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Authors
Cecile Leduc
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Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.
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Magnetic resonance imaging (MRI) is a non-invasive and label-free technique widely used in medical diagnosis and life science research, and its success has benefited greatly from continuing efforts on enhancing contrast and resolution. Here we reported nanoscale MRI in a single cell using an atomic-size quantum sensor. With nitrogen-vacancy center in diamond, the intracellular protein ferritin has been imaged with a spatial resolution of ~ 10 nanometers, and ferritin-containing organelles were co-localized by correlative MRI and electron microscopy. Comparing to the current micrometer resolution in current state-of-art conventional MRI, our approach represents a 100-fold enhancement, and paves the way for MRI of intracellular proteins.
We have developed a novel method to evaluate the potential profile of a molecular motor at each chemical state from only the probes trajectory and applied it to a rotary molecular motor F$_1$-ATPase. By using this method, we could also obtain the information regarding the mechanochemical coupling and energetics. We demonstrate that the position-dependent transition of the chemical states is the key feature for the highly efficient free-energy transduction by F$_1$-ATPase.
Single molecule localization microscopy (SMLM) techniques enable imaging biological samples well beyond the diffraction limit of light, but they vary significantly in their spatial and temporal resolutions. High-order statistical analysis of temporal fluctuations as in superresolution optical fluctuation imaging (SOFI) also enable imaging beyond diffraction limit, but usually at a lower resolution as compared to SMLM. Since the same data format is acquired for both methods, their algorithms can be applied to the same data set, and thus may be combined synergistically to improve overall imaging performance. Here, we find that SOFI converges much faster than SMLM, provides additive information to SMLM, and can efficiently reject background. We then show how SOFI-assisted SMLM imaging can improve SMLM image reconstruction by rejecting common sources of background, especially under low signal-to-background conditions. The performance of our approach was evaluated using a realistic simulation of fluorescence imaging we developed and further demonstrated on experimental SMLM images of the plasma membrane of activated fixed and live T cells. Our approach significantly enhances SMLM performance under demanding imaging conditions and could set an example for synergizing additional imaging techniques.
Voltage-gated sodium (Na$_mathrm{v}$) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Na$_mathrm{v}$ channel localization to the axon initial segment is vital to action potential initiation. Na$_mathrm{v}$ channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Na$_mathrm{v}$ channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Na$_mathrm{v}$1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Na$_mathrm{v}$1.6 channels localized to stable nanoclusters $sim$230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Na$_mathrm{v}$1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Na$_mathrm{v}$1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Na$_mathrm{v}$ channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Na$_mathrm{v}$1.6 was observed to be transiently trapped in the nanoclusters. Somatic Na$_mathrm{v}$1.6 nanoclusters represent a new, to our knowledge, type of Na$_mathrm{v}$ channel localization, and are hypothesized to be sites of localized channel regulation.
Surface enhanced Raman scattering (SERS) is optically sensitive and chemically specific to detect single molecule spectroscopic signatures. Facilitating this capability in optically-trapped nanoparticles at low laser power remains a significant challenge. In this letter, we show single molecule SERS signatures in reversible assemblies of trapped plasmonic nanoparticles using a single laser excitation (633 nm). Importantly, this trap is facilitated by the thermoplasmonic field of a single gold nanoparticle dropcasted on a glass surface. We employ bi-analyte SERS technique to ascertain the single molecule statistical signatures, and identify the critical parameters of the thermoplasmonic tweezer that provide this sensitivity. Furthermore, we show the utility of this low power ($approx$0.1 mW/$mu$m^2) tweezer platform to trap single gold nanoparticle and transport assembly of nanoparticles. Given that our configuration is based on a dropcasted gold nanoparticle, we envisage its utility to create reconfigurable plasmonic metafluids in physiological and catalytic environments, and can be potentially adapted as an in-vivo plasmonic tweezer.
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