Single-molecule tracking in living cells using single quantum dot applications

Abstract

Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes.

Keywords: in vivo real-time tracking.; membrane dynamics; neuroscience; nuclear pore complex; quantum dot; single molecule tracking.

Figure 1

Number of research report from 1985 to 2010 containing the key words (a) “single molecule tracking” and “quantum dot”, (b) “single molecule tracking”, and (c) “quantum dot”. The data are from WEB of KNOWLEDGE^SM by THOMSON REUTERS (2011).

Figure 2

Measurement of QD antibunching. (a) Photoluminescence intensity image of quantum dots via two-photon excitation at room temperature (scale bar 1 μm); (b) corresponding photoluminescence life time information image; and (c) coincidence measurement of PL photon pairs of a single quantum dot (arrow in (a)). Reprinted figure with permission from Matthias D. Wissert, Birgit Rudat, Uli Lemmer, and Hans-Jürgen Eisler, Quantum dots as single-photon sources: Antibunching via two-photon excitation, Fig. 3, Physical Review B Vol 83, 113304 (2011). Copyright (2011) by the American Physical Society. http://link.aps.org/doi/10.1103/PhysRevB.83.113304. Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or part, without prior written permission from the American Physical Society.

Figure 3

(A) Three-dimensional trajectory of a single QD-labeled IgE-FcεRI on an unstimulated mast cell at 37˚C. A rainbow color scheme is used to denote the passage of time. (B) Counts used for 3D tracking and feedback. (C) CCD image showing the receptor location relative to the mast cell (arrow points to the QD). (D) Z-position of this receptor, showing over 4 μm of Z-motion that would be missed in CCD-based tracking methods. (E) Mean-squared displacement (blue) and fit (red). The motion is highly compartmentalized. (F) Photon pair correlation measurement derived from this ~14 s long trajectory that shows fluorescence photon antibunching. Reprinted with permission from Nathan P. Wells, Guillaume A. Lessard, Peter M. Goodwin, Mary E. Phipps, Patrick J. Cutler, Diane S. Lidke, Bridget S. Wilson, and James H. Werner, Fig.3, Nano Letters Vol 10, 4732-4737 (2010). Copyright (2010) American Chemical Society.

Figure 4

Single QD tracking of Av-GPI by total internal reflection fluorescence (TIRF) microscopy. (A) First frame from a dual-color TIRF movie of a HeLa cell. Av-GPIs in the ventral plasma membrane are labeled with QDs (red) and GM1 molecules are labeled with Alexa-488 CT×B (green). (B) Selected frames from a region of interest (white square in (A)) in which diffusing Av-GPIs are tracked. Diffusion trajectories are determined by the series of fitted positions, connected by a straight line. Notice that Alexa-488 CT×B bleaches fast compared to QDs and the signal was nearly completely lost after 10 s. To facilitate visualization, the QD point-spread-function size was intentionally expanded. Tracking was performed on raw images. (C) Overlay of Av-GPI trajectories with the mean intensity projection image (ΣImean) for the Alexa-488 CT×B channel. This approach allows colocalization studies of Av-GPIs within fixed/slow diffusing GM1-rich domains despite the fast photobleaching of Alexa-488 CT×B. Reprinted with permission from Fabien Pinaud, Xavier Michalet, Gopal Iyer, Emmanuel Margeat, Hsiao-Ping Moore, and Shimon Weiss, Fig.2, Traffic Vol 10, 691-712 (2009). Copyright (2009) John Wiley and Sons.

Figure 5

Experimental design. (a) Diagram of a QD-based cargo. The snurportin-1 importin-β binding (IBB) /Z-domain fusion protein is coupled via a bifunctional succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate crosslinker (SMCC) to the amino-PEG polymer coat of a fluorescent QD. The three helix Z-domain acts as a spacer to correctly present the importin-β binding for biological function. Not to scale. (b) Dynamic light scattering size distributions of QD- IBB cargos in the presence and absence of IBB. (c) Dwell time distribution of all QD interactions with the nuclear pore complex. The time axis is truncated at 300 s. (d) Bright-field image of a nucleus with a QD fluorescence image (with background subtraction applied) overlaid in red. A single QD cargo at the nuclear envelope is boxed. (e) Individual consecutive frames from a single-molecule experiment showing the arrival (first frame) from the cytoplasm and departure (final frame) of the cargo into the nucleus. The centroids determined from fitting of the PSF (point spread function) are overlaid as red crosses. Frame numbers are in the bottom left hand corner of each frame. Movies were captured at 40 Hz. Reprinted with permission from Alan R. Lowe, Jake J. Siegel, Petr Kalab, Merek Siu, Karsten Weis, and Jan T. Liphardt, Fig.1, Nature Vol 467, 600-603 (2010). Copyright (2010) Nature Publishing Group. http://www.nature.com/nature/journal/v467/n7315/full/nature09285.html.